Constructs for gene expression analysis

ABSTRACT

The present invention relates generally to constructs and their use in gene expression or gene regulation assays. More particularly, the present invention provides expression vectors and/or reporter vectors providing kinetics of protein expression with improved temporal correlation to promoter activity. Even more particularly, the invention provides expression vectors comprising a transcribable polynucleotide which comprises a sequence of nucleotides encoding a RNA element that modulates the stability of a transcript corresponding to the transcribable polynucleotide. The present invention provides, inter alia, novel vectors, useful for identifying and analysing cis- and trans-acting regulatory sequences/factors as well as vectors and genetically modified cell lines or organisms that are particularly useful for drug screening and drug discovery.

This application is a continuation of application Ser. No. 10/658,093,filed Sep. 9, 2003, which is a continuation-in-part of InternationalApplication No. PCT/AU02/00351, filed Mar. 8, 2002, which was publishedin English and claims priority under § 119(e) to Provisional ApplicationNo. 60/274,770, filed Mar. 9, 2001.

BACKGROUND OF THE INVENTION

This invention relates generally to constructs and their use in geneexpression or gene regulation assays. More particularly, the presentinvention provides expression vectors and/or reporter vectors providingkinetics of protein expression with improved temporal correlation topromoter activity. The present invention provides, inter alia, novelvectors and cell lines useful for modulating gene expression,identifying and analysing regulatory sequences, new targets and reagentsfor therapeutic intervention in human diseases and for drug-screening.

Bibliographic details of the publications referred to by author in thisspecification are collected at the end of the description.

The rapidly increasing sophistication of recombinant DNA technology isgreatly facilitating research and development in the medical and alliedhealth fields. A particularly important area of research is the use ofexpression vectors to study gene expression. However, until now, areal-time analysis of gene expression has been limited by the lack ofsuitably designed vectors.

Reporter assays permit an understanding of what controls the expressionof a gene of interest e.g., DNA sequences, transcription factors, RNAsequences, RNA-binding proteins, signal transduction pathways andspecific stimuli.

Furthermore, reporter assays can be used to identify aspects of generegulation that serve as new targets for therapeutic intervention inhuman disease. Reporter assays can potentially be used to screen drugsfor their ability to modify gene expression. However, the cost and timerequired for current reporter assay systems, together with theinaccuracies caused by the lengthy response times, has limited thisapplication.

Genomic sequences have promoter sequences, generally upstream of thecoding region, which dictate the cell specificity and inducibility oftranscription and thereby affect the level of expression of proteinproducts.

Specific sequence elements, typically rich in the nucleotide bases A andU and often located in the 3′-UTR of a gene, affect the stability of themRNA and thereby affect the level of expression of the protein product.RNA-binding proteins bind certain mRNA sequences and thereby regulatemRNA stability and protein expression. Other sequences and trans-actingproteins modulate other post-transcriptional pathways such astranslational efficiency, mRNA splicing and mRNA export into thecytoplasm.

A common application of gene reporter assays is the study of DNAsequences that regulate transcription. Typically, these sequences arelocated in the promoter region, 5′ of the transcription start site. SuchDNA elements are tested by cloning them into a similar site within areporter plasmid, such that they drive and/or regulate transcription andtherefore, expression of reporter protein. The reporter protein shouldbe distinguishable from endogenous proteins and easily quantified.Various reporter proteins are used, the most common being luciferase,chloramphenicol transferase (CAT) and β galactosidase (β-gal).

The reporter protein is quantified in an appropriate assay and oftenexpressed relative to the level of a control reporter driven by aubiquitous promoter such as for example the promoter SV40. The controlreporter must be distinguishable from the test reporter and is containedon a separate vector that is co-transfected with the test vector andused to control for transfection efficiency. Such assays are based onthe premise that cells take up proportionally equal amounts of bothvectors. Transient transfections of plasmid vectors are most commonlyused.

The assays described above are used to identify a promoter region or thespecific elements within a promoter. Alternatively, they are used tostudy the response to various stimuli of a promoter or regulatoryelement. In some applications, the reporter constructs, or thetransfected cells, are placed into an organism to study promoterfunction in vivo.

Another application of these reporter assays is the study or measurementof signal transduction pathways upstream of a specific promoter. Forexample, a promoter dependent on mitogen activated protein kinase (MAPK)for transcription can be linked to a reporter construct and used tomeasure the level of MAPK activation (or MAPK-dependent transcription)in cells. This technique can be utilized with a variety of informativepromoters or enhancers and can be applied to cells or living organismssuch as transgenic mice. For example, a photon camera can be used tomeasure luciferase reporter activity in whole mice containing aluciferase reporter linked to a promoter of interest (Contag, et al,1997).

Luciferase is the most commonly used reporter assay for in vitrosystems. The Dual Luciferase assay (DLA; Promega, Madison, Wis., USA),is an improvement over other luciferase based systems in that both testand control reporter can essentially be measured in the same assay. Asan example of current use, a typical DLA protocol is provided asfollows:

The putative promoter element is cloned upstream of a firefly luciferasereporter gene such that it drives its expression. This plasmid istransiently transfected into a cell line, along with a control plasmidcontaining the Renilla luciferase gene driven by the SV40 promoter.˜2-50% of cells take up plasmid and express the reporters for ˜3 days.The kinetics of expression involve an increase during the first ˜24 h asluciferase protein accumulates, followed by a decrease from ˜48 h as thenumber of plasmids maintained within the cells declines. 24-48 h aftertransfection, cells are harvested and lysed. Cell lysates are incubatedwith substrates specific to firefly luciferase and activity (lightemission) is measured using a luminometer (96 well plate or individualsamples). Additional substrates are then added, which inactivate fireflyluciferase but allow Renilla luciferase to generate light. Renillaluciferase activity can then be measured.

The level of firefly luciferase activity is dependent, not only onpromoter activity, but also on transfection efficiency. This variesgreatly, depending on the amount of DNA, the quality of the DNApreparation and the condition of the cells. The co-transfected controlplasmid (Renilla luciferase driven by the SV40 promoter) is used tocorrect for these variables, based on the premise that Renillaluciferase activity is proportional to the amount of firefly luciferaseplasmid taken up by the cells. Data are expressed as firefly luciferaseactivity/Renilla luciferase activity.

The disadvantages of the Dual Luciferase assay are as follows:

-   -   (i) Reagents are expensive and perishable and must be freshly        prepared.    -   (ii) Generally this assay involves the preparation of cell        lysates, which is time consuming and adds inaccuracy. e.g., loss        of cells during lysis, pipetting errors, residual buffer/medium        altering volumes.    -   (iii) Each sample yields only one datum point being the total        activity of the cell population. No information is gained        concerning the percentage of cells that express the reporter,        nor the amount of expression per cell.    -   (iv) The transfection control (Renilla) does not always correct        for huge variation in transfection efficiencies because:        -   (a) Certain DNA preparations transfect/express poorly            (perhaps due to reduced proportion of supercoiled DNA), but            do not cause a corresponding decrease in the amount of            co-transfected control plasmid.        -   (b) There is evidence of cross-talk between the promoters of            the two plasmids, such that control reporter activity is            dependent on the construct with which it is co-transfected,            e.g., expression of Renilla luciferase seems highest when            co-transfected with a plasmid containing a strong promoter.            Interference between promoters has also limited, if not            prevented, the use of single plasmids expressing both test            and control reporters.        -   (c) A common application of both transcriptional and            post-transcriptional studies is to measure            activation/suppression by various stimuli (e.g., PMA, EGF,            hormones). Unfortunately, SV40, RSV, TK and probably many            other ubiquitously expressed promoters are activated by a            variety of stimuli. Since these promoters are used to drive            expression of the transfection control reporter (Renilla),            these reporters do not give a true reflection of            transfection efficiency following such treatments. (Ibrahim            et al. 2000).        -   (d) Differences in the half-lives of firefly vs Renilla            luciferase proteins and perhaps mRNAs make the whole system            very time-sensitive.        -   (e) Rapidly diminishing light emission, particularly for            Renilla luciferase, require absolute precision in the timing            of measurement.        -   (f) The relatively long half-lives of luciferase proteins            and mRNAs effectively mask temporal changes in transcription            (e.g., following various stimuli or treatments).

In existing post-transcriptional/mRNA stability reporter assays,candidate elements, thought to affect mRNA stability are cloned into thecorresponding region of a reporter vector (e.g., firefly luciferase)driven by a constitutive promoter such as SV40 or RSV. Changes inexpression relative to the empty vector (same vector without element ofinterest) are assumed to be the result of altered mRNA stability ortranslational efficiency. More complex assays are required todistinguish the two possibilities. As with the preliminary describedtransfection assays, a transfection control plasmid (e.g., Renillaluciferase driven by a constitutive promoter such as SV40 or RSV) isco-transfected to allow correction for transfection efficiency. Theseassays suffer from the following additional disadvantages:

-   -   (1) Existing vectors were not designed for post-transcriptional        studies and have no means for switching off transcription.    -   (2) The purpose of these protocols is to study the        post-transcriptional effects of candidate mRNA elements.        However, these elements can also affect transcription of the        reporter at the level of DNA. Furthermore, since the endogenous        promoter of the gene of interest is not used, any        transcriptional effects seen may have little physiological        relevance.

Other systems for studying mRNA stability exist but involve directmeasurement of the mRNA rather than a protein reporter. Due to thelabour-intensive nature of protocols for quantifying mRNA, such systemsare far more time consuming.

One system, for example, utilizes the c-fos promoter, which responds toserum induction with a brief burst of transcription. Putativeinstability elements are cloned into the 3-UTR of a Beta Globin (BBB)construct, which expresses the very stable beta globin mRNA under thecontrol of a serum-inducible (c-fos) promoter. Transfected cells(generally NIH 3T3 cells) are first serum starved and then exposed tomedium containing serum. The brief nature of the transcriptionalresponse allows the kinetics of reporter mRNA degradation to be followedin a time course. This assay suffers from the following disadvantages:

-   -   (i) Quantifying mRNA rather than reporter protein is very time        consuming and is therefore not applicable to rapid screening.    -   (ii) Can only be used in cells that support serum inducibility        of the c-fos promoter. For example, many tumour cell lines        maintain c-fos promoter activity in the absence of serum.    -   (iii) In cells such as NIH 3T3 cells, which do have the desired        serum response, serum deprivation causes a cell cycle block and        subsequent addition of serum, releases the cells from this block        in a synchronous manner. Therefore, mRNA stability can only be        measured in specific stages of the cell cycle.    -   (iv) In addition to activating the c-fos promoter, serum        activates a multitude of other pathways, which introduce        unwanted variables and prevent the study of more specific        stimuli.

In another assay, cells are treated with drugs, such as Actinomycin Dthat inhibit transcription from all genes. The mRNA levels are measuredin a time course to determine mRNA degradation rates. This system isused to study endogenous genes and suffers from the followingdisadvantages:

-   -   (i) Transcriptional inhibitors are extremely toxic at doses        required such that mRNA stability is often being measured in        stressed or dying cells.    -   (ii) Transcription inhibitors possess numerous unwanted        activities including stabilization of certain mRNAs.    -   (iii) The process blocks transcription from all genes such that        many signal transduction cascades are blocked, whereas others        are activated. Therefore, results may not be physiologically        relevant.    -   (iv) The technique is extremely labour intensive.    -   (v) The technique is highly variable within and between assays.    -   (vi) The technique is often not sensitive enough for transient        transfection reporter assays, particularly in cells with low        transfection efficiency.

There is a need therefore to develop improved vectors and systems forconducting gene expression assays and in particular post-transcriptionalassays as well as assays that permit a more real-time determination ofchanges in gene expression.

SUMMARY OF THE INVENTION

The present invention is predicated in part on the development of anovel series of constructs and methods which permit inter aliamodulation and determination of transcript stability and/or improvedreal-time determination of gene expression.

Accordingly, in one aspect of the present invention constructs areprovided for assaying the activity of gene expression-modulatingelements (e.g., transcriptional control elements and cis-actingregulatory elements) or for identifying elements of this type or agentsthat modulate their activity. These constructs generally comprise inoperable linkage: a polynucleotide that encodes a polypeptide and anucleic acid sequence that encodes a RNA element that modulates thestability of a transcript encoded by the polynucleotide. In someembodiments, the polypeptide has an intracellular half-life of less thanabout 1, 2 or 3 hours.

In some embodiments, the RNA element is a destabilising element thatreduces the stability of the transcript. Suitably, in these embodiments,the nucleic acid sequence is, or is derived from, a gene selected fromc-fos, c-jun, c-myc, GM-CSF, IL-3, TNF-alpha, IL-2, IL-6, IL-8, IL-10,Urokinase, bcl-2, SGLT1 (Na(+)-coupled glucose transporter), Cox-2(cyclooxygenase 2);. IL-8, PAI-2 (plasminogen activator inhibitor type2), beta1-adrenergic receptor or GAP43. Illustrative examples of suchnucleic acid sequences include, but are not limited to, the nucleotidesequences set forth in SEQ ID NOS 1 to 23, especially in SEQ ID NO:1,13, 19 or 49, or biologically active fragments thereof, or variants orderivatives of these.

In other embodiments, the RNA element is a stabilising element thatincreases the stability of the transcript. Suitably, in theseembodiments, the nucleic acid sequence is, or is derived from, a geneselected from alpha2 globin, alpha1 globin, beta globin, growth hormone,erythropoietin, ribonucleotide reductase R1 or m1 muscarinicacetylcholine.

In some embodiments, the polynucleotide and the nucleic acid sequenceare heterologous to each other.

In some embodiments, the polypeptide comprises a protein-destabilisingelement, which is suitably selected from a PEST sequence, an ubiquitin,a biologically active fragment of an ubiquitin, or variant or derivativeof these.

In some embodiments, the polypeptide is a reporter protein, which issuitably selected from an enzymatic protein or a protein associated withthe emission of light (e.g., a fluorescent or luminescent protein).Illustrative examples of suitable reporter proteins include, but are notlimited to, Luciferase, GFP, SEAP, CAT, or biologically active fragmentsthereof, or variants or derivatives of these. In other embodiments, thepolypeptide is a protein having at least a light-emitting activity and aselection marker activity. In these embodiments, the polypeptide issuitably encoded by a chimeric gene which includes a coding sequencefrom a gene encoding a light-emitting protein and a coding sequence froma gene encoding a selectable marker protein. In certain embodiments, thelight-emitting protein is selected from Green Fluorescent Protein,Luciferase and their biologically active fragments, variants andderivatives and the selectable marker protein is selected from kanamycinkinase, neomycin phosphotransferase, aminoglycoside phosphotransferase,puromycin N-acetyl transferase, puromycin resistance protein and theirbiologically active fragments, variants and derivatives.

In some embodiments, the constructs further comprise one or more of thefollowing: a transcriptional control element for regulating expressionof the polynucleotide and of the nucleic acid sequence; a cis-actingregulatory element (e.g., a transcriptional enhancer) for modulating theactivity of the transcriptional control element; a reporter gene; atleast one cloning site for introducing a sequence of nucleotides; apolyadenylation sequence; a selectable marker; an origin of replication;a translation modulating element (e.g., a translational enhancer) formodulating translation of a transcript encoded by the polynucleotide andan intron or other post-transcriptional regulatory element (e.g.,woodchuck post-transcriptional regulatory element from woodchuckhepatitis virus, which is an example of a mRNA nuclear export signal)for modulating other aspects of post-transcriptional gene regulation. Incertain illustrative examples, the constructs comprise in operablelinkage: a polynucleotide that encodes a polypeptide and a nucleic acidsequence that encodes a RNA element that modulates the stability of atranscript encoded by the polynucleotide, wherein the construct lacks,but comprises a site for introducing, a gene expression-modulatingelement in operable connection with the polynucleotide and the nucleicacid sequence. In other illustrative examples, the constructs comprise agene expression-modulating element in operable linkage with apolynucleotide that encodes a polypeptide and with a nucleic acidsequence that encodes a RNA element that modulates the stability of atranscript encoded by the polynucleotide, wherein the construct furthercomprises a site for introducing a post-transcriptional control element.In these examples, the polypeptide desirably has an intracellularhalf-life of less than about 1, 2 or 3 hours and more desirablycomprises a protein-destabilising element.

In embodiments in which a construct comprises a cloning site, thecloning site is suitably selected from a multiple cloning site or a sitethat is cleavable enzymatically, chemically or otherwise to provide alinearised vector into which PCR amplification products are clonabledirectly.

The constructs are typically in the form of a vector. In someembodiments, the constructs are suitable for assaying the activity of atranscriptional control element or the activity of a cis-actingregulatory element or both.

In a related aspect, the present invention provides constructs forassaying the activity of gene expression-modulating elements (e.g.,transcriptional control elements and cis-acting regulatory elements) orfor identifying elements of this type or agents that modulate theiractivity. These constructs generally comprise in operable linkage: apolynucleotide that encodes a polypeptide comprising aprotein-destabilising element, and a nucleic acid sequence that encodesa RNA element that modulates the stability of a transcript encoded bythe polynucleotide. In some embodiments, the polypeptide is a reporterprotein.

In another aspect, the present invention provides a cell comprising oneor more constructs as broadly described above. Typically, the cell isselected from prokaryotic (e.g., bacterial) or eukaryotic cells (e.g.,mammalian including human cells).

In yet another aspect, the present invention provides a geneticallymodified non-human organism comprising one or more constructs as broadlydescribed above.

Still another aspect of the present invention provides methods forassaying the activity of a transcriptional control element. Thesemethods generally comprise: (1) expressing from the transcriptionalcontrol element a polynucleotide that encodes a polypeptide and that isoperably connected to a nucleic acid sequence that encodes a RNA elementthat modulates the stability of a transcript encoded by thepolynucleotide; and (2) measuring the level or functional activity ofthe polypeptide produced from the expression. In some embodiments, theexpression of the polynucleotide is carried out in the presence andabsence of a test agent. In these embodiments, the methods furthercomprise comparing the level or functional activity of the polypeptideproduced in the presence and absence of the test agent. Suitably, theexpression of the polynucleotide is carried out in a first cell type orcondition and in a second cell type or condition, wherein a differencein the level or functional activity of the polypeptide in the presenceof the test agent between the cell types or conditions providesinformation on the effect of the test agent on those cell types orconditions (e.g., mode of action or specificity). In some embodiments,the activity of the transcriptional control element is a measure of acellular event, which includes but is not limited to cell cycleprogression, apoptosis, immune function, modulation of a signaltransduction pathway, modulation of a regulatory pathway, modulation ofa biosynthetic pathway, toxic response, cell differentiation and cellproliferation.

In some embodiments, the methods comprise: (1) expressing from a firsttranscriptional control element in a first construct a firstpolynucleotide that encodes a first polypeptide and that is operablyconnected to a nucleic acid sequence that encodes a RNA element thatmodulates the stability of a transcript encoded by the firstpolynucleotide; (2) measuring the level or functional activity of thefirst polypeptide produced from the first construct; (3) expressing froma second transcriptional control element in a second construct a secondpolynucleotide that encodes a second polypeptide and that is operablyconnected to a nucleic acid sequence that encodes a RNA element thatmodulates the stability of a transcript encoded by the secondpolynucleotide, wherein the expression of the second polynucleotide iscarried out in the presence or absence of the test agent, and whereinthe second transcriptional control element is different than the firsttranscriptional control element; (4) measuring the level or functionalactivity of the second polypeptide produced from the second construct;and (5) comparing the level or functional activity of the secondpolypeptide with the level or functional activity of the firstpolypeptide in the presence or absence of the test agent. The firstconstruct and the second construct may be in the form of separateconstructs or a single chimeric construct. For example, the first andsecond constructs may be present on the same vector or on separatevectors. Desirably, the first polypeptide and the second polypeptide aredetectably distinguishable. The first construct and the second constructmay be contained within a single cell or within different cells. In someembodiments, at least one of the first and second polypeptides has anintracellular half-life of less than about 1, 2 or 3 hours.

Still another aspect of the present invention provides methods foridentifying an agent that modulates the activity of a geneexpression-modulating element (e.g., transcriptional control elementsand cis-acting regulatory elements). These methods generally comprise:(a) expressing under the control of the gene expression-modulatingelement a polynucleotide that encodes a polypeptide and a nucleic acidsequence that encodes a RNA element that modulates the stability of atranscript encoded by the polynucleotide in the presence and absence ofa test agent; (b) measuring the level or functional activity of thepolypeptide in the presence and absence of the test agent; and (c)comparing those levels or functional activities, wherein a differencebetween the level or functional activity of the polypeptide in thepresence and absence of the test agent indicates that the test agentmodulates the activity of the gene expression-modulating element. Insome embodiments, the polypeptide comprises a protein-destabilisingelement. Suitably, the polypeptide has an intracellular half-life ofless than about 1, 2 or 3 hours. In some embodiments, the polypeptide isa reporter protein.

In yet another aspect, the present invention provides constructs foridentifying or assaying the activity of a cis-acting regulatory element.These constructs generally comprise a transcriptional control element inoperable linkage with: a polynucleotide that encodes a polypeptide and anucleic acid sequence that encodes a RNA element that modulates thestability of a transcript encoded by the polynucleotide, wherein theconstructs further comprise a site for introducing cis-acting regulatoryelement or a nucleotide sequence suspecting of being a cis-actingregulatory element in operable linkage with the transcriptional controlelement. In illustrative examples, the constructs lack, but comprise asite for introducing, a cis-acting regulatory element in said operablelinkage. In some embodiments, the transcriptional control element is aminimal promoter. In some embodiments of this type, the polypeptidecomprises a protein-destabilising element. Suitably, the polypeptide hasan intracellular half-life of less than about 1, 2 or 3 hours. In someembodiments, the polypeptide is a reporter protein. In some embodiments,the activity of the cis-acting regulatory element is a measure of acellular event, which includes but is not limited to cell cycleprogression, apoptosis, immune function, modulation of a signaltransduction pathway, modulation of a regulatory pathway, modulation ofa biosynthetic pathway, toxic response, cell differentiation and cellproliferation.

A further aspect of the present invention provides methods for assayingthe activity of a post-transcriptional control element. These methodsgenerally comprise: (1) expressing from a transcriptional controlelement a polynucleotide that encodes a polypeptide and that is operablylinked to: a nucleic acid sequence that encodes the post-transcriptionalcontrol element; and (2) measuring the level or functional activity ofthe polypeptide produced from the expression. In some embodiments, thepolypeptide comprises a protein-destabilising element. Suitably, thepolypeptide has an intracellular half-life of less than about 1, 2 or 3hours. In some embodiments, the polypeptide is a reporter protein. Insome embodiments, the expression of the polynucleotide is carried out inthe presence and absence of a test agent. In these embodiments, themethods desirably further comprise comparing the level or functionalactivity of the polypeptide produced in the presence and absence of thetest agent. In some of these embodiments, the expression of thepolynucleotide is carried out in a first cell type or condition and in asecond cell type or condition, wherein a difference in the level orfunctional activity of the polypeptide in the presence of the test agentbetween the cell types or conditions provides information on the effectof the test agent on those cell types or conditions (e.g., mode ofaction or specificity). In some embodiments, the activity of thepost-transcriptional control element is a measure of a cellular event,which includes but is not limited to cell cycle progression, apoptosis,immune function, modulation of a signal transduction pathway, modulationof a regulatory pathway, modulation of a biosynthetic pathway, toxicresponse, cell differentiation and cell proliferation.

In some embodiments, the methods comprise: (a) expressing from a firsttranscriptional control element in a first construct a firstpolynucleotide that encodes a first polypeptide and that is operablylinked to: a nucleic acid sequence that encodes the post-transcriptionalcontrol element, wherein the expression of the first polynucleotide isoptionally carried out in the presence or absence of a test agent; (b)measuring the level or functional activity of the first polypeptideproduced from the first construct; (c) expressing from a secondtranscriptional control element in a second construct a secondpolynucleotide, which encodes a second polypeptide but which is notoperably linked to the nucleic acid sequence that encodes thepost-transcriptional control element, wherein the second polypeptide isthe same as, or different than, the first polypeptide, wherein thesecond transcriptional control element is the same as, or differentthan, the first transcriptional control element and wherein theexpression of the second polynucleotide is optionally carried out in thepresence or absence of the test agent; (d) measuring the level orfunctional activity of the second polypeptide produced from the secondconstruct; and (e) comparing the level or functional activity of thesecond polypeptide with the level or functional activity of the firstpolypeptide optionally in the presence or absence of the test agent. Inthese embodiments, the first construct and the second construct may bein the form of separate constructs or a single chimeric construct. Forexample, the first and second constructs may be present on the samevector or on separate vectors. The first and second constructs may becontained within a single cell or within different cells. In certainembodiments, the first polypeptide and the second polypeptide aredetectably distinguishable. Suitably, at least one of the first andsecond polypeptides has an intracellular half-life of less than about 1,2 or 3 hours. In some embodiments of this type, one or both of the firstand second polypeptides comprise(s) a protein-destabilising element. Insome embodiments, the transcriptional control element is modulatable,including inducible or repressible promoters. In these embodiments, themethods desirably further comprise (1) inducing or repressing the firstor second transcriptional control element; and (2) measuring changes inthe level or functional activity of the first or second polypeptide overtime.

In still a further aspect, the present invention provides methods foridentifying a nucleotide sequence that encodes a post-transcriptionalcontrol element that modulates the expression of a RNA transcript from afirst polynucleotide that encodes a polypeptide. These methods generallycomprise: (i) expressing from a first transcriptional control element ina first construct the first polynucleotide, which is operably connectedto a test nucleotide sequence suspected of encoding thepost-transcriptional control element; (ii) expressing from a secondtranscriptional control element in a second construct a secondpolynucleotide, which encodes a second polypeptide, but which is notoperably connected to the test nucleotide sequence, wherein the secondpolypeptide is the same as, or different than, the first polypeptide andwherein the second transcriptional control element is the same as, ordifferent than, the first transcriptional control element; and (iii)comparing the level or functional activity of the polypeptides from thefirst and second constructs, wherein a difference between the level orfunctional activity of the first polypeptide and the level or functionalactivity of the second polypeptide indicates that the test nucleotidesequence encodes a post-transcriptional control element. In someembodiments, at least one of the first and second polypeptides has anintracellular half-life of less than about 1, 2 or 3 hours. In someembodiments, at least one of the first and second polypeptides comprisesa protein-destabilising element. In some embodiments, the first andsecond polypeptides are reporter proteins. In some embodiments, thetranscriptional control element is modulatable, including inducible orrepressible promoters. In these embodiments, the methods desirablyfurther comprise (1) inducing or repressing the first or secondtranscriptional control element; and (2) measuring changes in the levelor functional activity of the first or second polypeptide over time.

In still another aspect, the present invention provides methods foridentifying an agent that modulates the activity of apost-transcriptional control element that modulates the expression of aRNA transcript from a polynucleotide that encodes a polypeptide. Thesemethods generally comprise: (a) expressing from a transcriptionalcontrol element the polynucleotide, which is operably connected to anucleic acid sequence that encodes the post-transcriptional controlelement, wherein the expression of the polynucleotide is carried out inthe presence and absence of a test agent; (b) measuring the level orfunctional activity of the polypeptide in the presence and absence ofthe test agent; and (c) comparing those levels or functional activities,wherein a difference between the level or functional activity of thepolypeptide in the presence and absence of the test agent indicates thatthe test agent modulates the activity of the post-transcriptionalcontrol element. In some embodiments, the expression of thepolynucleotide is carried out in a first cell type or condition and in asecond cell type or condition, wherein a difference in the level orfunctional activity of the polypeptide in the presence of the test agentbetween the cell types or conditions provides information on the effectof the test agent on those cell types or conditions (e.g., mode ofaction or specificity). In some embodiments, the polypeptide comprises aprotein-destabilising element. Suitably, the polypeptide has anintracellular half-life of less than about 1, 2 or 3 hours. In someembodiments, the polypeptide is a reporter protein. In some embodiments,the transcriptional control element is modulatable, including inducibleor repressible promoters. In these embodiments, the methods desirablyfurther comprise (1) inducing or repressing the first or secondtranscriptional control element; and (2) measuring changes in the levelor functional activity of the first or second polypeptide over time.

In some embodiments, these methods comprise (i) expressing from a firsttranscriptional control element a first polynucleotide, which encodes afirst polypeptide and which is operably connected to a nucleic acidsequence that encodes the post-transcriptional control element, whereinthe expression of the first polynucleotide is carried out in thepresence and absence of a test agent; (ii) measuring the level orfunctional activity of the polypeptide in the presence and absence ofthe test agent; (iii) expressing from a second transcriptional controlelement in a second construct a second polynucleotide, which encodes asecond polypeptide, but which is not operably connected to the nucleicacid sequence, wherein the second polypeptide is the same as, ordifferent than, the first polypeptide, wherein the secondtranscriptional control element is the same as, or different than, thefirst transcriptional control element and wherein the expression of thesecond polynucleotide is carried out in the presence or absence of thetest agent; (iv) measuring the level or functional activity of thesecond polypeptide from the second construct in the presence or absenceof the test agent; and (v) comparing the level or functional activity ofthe second polypeptide with the level or functional activity of thefirst polypeptide in the presence or absence of the test agent. In theseembodiments, the first construct and the second construct may be in theform of separate constructs or a single chimeric construct. For example,the first and second constructs may be present on the same vector or onseparate vectors. The first and second constructs may be containedwithin a single cell or within different cells. In some embodiments, atleast one of the first and second polypeptides comprises aprotein-destabilising element. Suitably, one or both of the first andsecond polypeptides has/have an intracellular half-life of less thanabout 1, 2 or 3 hours. Typically, the first polypeptide and the secondpolypeptide are detectably distinguishable. In some embodiments, thefirst and second polynucleotides are transcribed from the sametranscriptional control element, illustrative examples of which includea bi-directional promoter.

In some embodiments, the transcriptional control element is modulatable.For example, the transcriptional control element may be repressible(e.g., a TRE or derivative thereof) or inducible. In these embodiments,the methods further comprise (A) inducing or repressing the firsttranscriptional control element; and (B) measuring a change in the levelor functional activity of the polypeptide over time.

Still another aspect of the present invention provides constructs foridentifying or assaying the activity of a post-transcriptional controlelement that modulates the expression of a transcript. These constructsgenerally comprise a transcriptional control element that is operablyconnected to: a polynucleotide from which the transcript is transcribedand which encodes a polypeptide having an intracellular half-life ofless than about 3 hours; and a cloning site for introducing a nucleotidesequence that encodes, or is suspected to encode, thepost-transcriptional control element. Suitably, the polypeptide has anintracellular half-life of less than about 1 or 2 hours. In someembodiments of this type, the polypeptide comprises aprotein-destabilising element. In some embodiments, the polypeptide is areporter protein.

Yet another aspect of the present invention provides constructs foridentifying or assaying the activity of a post-transcriptional controlelement that modulates the expression of a transcript. These constructsgenerally comprise a transcriptional control element that is operablyconnected to: a polynucleotide from which the transcript is transcribedand which encodes a polypeptide comprising a protein-destabilisingelement; and a cloning site for introducing a nucleotide sequence thatencodes, or is suspected to encode, the post-transcriptional controlelement. Suitably, the polypeptide has an intracellular half-life ofless than about 1, 2 or 3 hours. In some embodiments, the polypeptide isa reporter protein.

Another aspect of the present invention provides methods for identifyinga transcriptional control element. These methods generally comprise: (1)subjecting a construct to conditions sufficient for RNA and proteinsynthesis to occur, wherein the construct comprises in operable linkage:a nucleotide sequence suspected of having transcriptional controlactivity; a polynucleotide that encodes a polypeptide and a nucleic acidsequence that encodes a RNA element that modulates the stability of atranscript encoded by the polynucleotide; and (2) detecting thepolypeptide produced from the construct. In some embodiments, thepolypeptide comprises a protein-destabilising element. Suitably, thepolypeptide has an intracellular half-life of less than about 1, 2 or 3hours.

Yet another aspect of the present invention provides methods foridentifying a cis-acting regulatory element that modulates the activityof a transcriptional control element. These methods generally comprise:(1) subjecting a construct to conditions sufficient for RNA and proteinsynthesis to occur, wherein the construct comprises in operable linkage:a nucleotide sequence suspected of having cis-acting regulatoryactivity; the transcriptional control element; a polynucleotide thatencodes a polypeptide and a nucleic acid sequence that encodes a RNAelement that modulates the stability of a transcript encoded by thepolynucleotide; and (2) detecting the polypeptide produced from theconstruct. In some embodiments, the polypeptide comprises aprotein-destabilising element. Suitably, the polypeptide has anintracellular half-life of less than about 1, 2 or 3 hours. In someembodiments, the polypeptide is a reporter protein.

In a further aspect, the present invention provides methods for assayingthe activity of a transcriptional control element. These methodsgenerally comprise: (i) expressing from the transcriptional controlelement a polynucleotide that encodes a polypeptide comprising aprotein-destabilising element and that is operably connected to anucleic acid sequence that encodes a RNA element that modulates thestability of a transcript encoded by the polynucleotide; and (ii)measuring the level or functional activity of the polypeptide producedfrom the construct.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of expression vectors encoding adestabilising mRNA.

FIG. 2 is a schematic representation of transcription reporter vectors;FIG. 2 a shows vector series 2; FIG. 2 b shows vector series 3 and FIG.2 c shows vector series 4.

FIG. 3 is a schematic representation of Bi-directional transcriptionreporter vectors; FIG. 3 a shows vector series 5 and FIG. 3 b showsvector series 6.

FIG. 4 is a schematic representation of reporter vectors for studyingpost-transcriptional regulation; FIG. 4 a shows vector series 7 and FIG.4 b shows vector series 8.

FIG. 5 is a graphical representation showing reporter activity as afunction of the amount of DNA transfected. A single DNA preparation of aplasmid encoding firefly luciferase was mixed at a 30:1 ratio with aseparate plasmid encoding Renilla luciferase. Both DNA preparationsappeared normal in spectrophotometry (OD260/280) and on ethidium bromidestained agarose gels (data not shown). Different volumes of this mixturewere transfected into cells such that the total quantity of DNA was 1, 2or 3 micrograms but the ratio of firefly to Renilla plasmids remainedthe same. Specifically, FIG. 5A shows that Renilla luciferase activitywas dependent on the amount of DNA transfected. However, fireflyluciferase activity (as shown in FIG. 5B) did not increase withincreasing amounts of DNA, perhaps because the firefly DNA preparationwas of poor quality. Consequently, the firefly/Renilla ratio (as shownin FIG. 5C), which would typically be used as a measure of the fireflypromoter activity, varied considerably depending on the amount of DNAused. These data demonstrate that co-transfections with Renilla plasmidsdo not adequately control for the transfection efficiency of the fireflyplasmid.

FIG. 6 is a graphical representation showing reporter activity forvarious promoter systems using the Dual Luciferase Assay. Six differentpromoter fragments (numbered 1-6) were cloned into pGL3 fireflyluciferase plasmids. One microgram of each clone was co-transfected with30 ng of Renilla (transfection control) plasmid, driven by an SV40promoter. Firefly and Renilla luciferase activities were measured usingthe Dual Luciferase Assay (Promega, Madison, Wis., USA). Results areexpressed as Renilla luciferase activity (A), Firefly luciferaseactivity (B) and firefly divided by Renilla activity (C). Similarresults were seen in multiple experiments using at least 2 differentpreparations of each construct. Renilla luciferase activity (as shown inFIG. 6A) is intended as a transfection control and analysis of thisresult alone would suggest an unusually high variation in transfectionefficiency. For example, Renilla luciferase activity is 3.5 fold higherwhen co-transfected with construct 4 compared to co-transfection withconstruct 3. Variations in DNA quality or errors in the quantificationof DNA seem unlikely as sources of error since the same pattern was seenwith a separate set of DNA preparations (data not shown). Fireflyluciferase activity (as shown in FIG. 6B) is influenced by bothtransfection efficiency and differences between promoters 1-6. Thepattern of differences is similar to that seen with Renilla (FIG. 6A).For example, 3 and 6 are low whilst 4 and 5 are high. However thesedifferences between constructs are more .marked with firefly (e.g.,construct 4 is 12 fold higher than construct 3), suggesting that theactivity of promoters 1-6 is somehow affecting expression of Renilla (orvice versa). Firefly/Renilla (FIG. 6C) is considered to be a measure oftrue firefly promoter activity (16) after correction for transfectionefficiency (Renilla). Again a similar pattern is seen, suggesting thatindeed 3 and 6 are the weakest promoters whilst 4 and 5 are thestrongest. Whilst it is possible that promoter activity (FIG. 6C)coincidentally correlated with transfection efficiency (FIG. 6A), thispossibility seems extremely unlikely given that similar results wereobtained with numerous different constructs and multiple differentpreparations of the same construct. It seems more likely that the levelof expression of Renilla luciferase is affected by the strength of thepromoter construct with which it is co-transfected. Consequently,apparent differences between promoters 1-6 are likely to be anunderestimation of the true differences.

FIG. 7 is a graphical representation showing different reporter levelsfor BTL, BTG2, BTG1 and BTG1N4 expression vectors on a time course afterblocking transcription. Tet-Off HeLa cells were transfected with thefollowing reporter plasmids, each containing a TRE promoter linked to areporter gene; BTL (luciferase), BTG2 (d2EGFP), BTG1 (d1EGFP) and BTG1N4(same as BTG1 but with 4 copies of the nonamer UUAUUUAUU [SEQ ID NO:1]present in the 3′-UTR-encoding region). Ten hrs after transfection, eachflask of cells was split into multiple small plates. Doxycycline (1μg/mL) was added at 24 hrs after transfection (time zero) to blocktranscription of the reporter genes. Reporter levels (fluorescence orluminescence) were measured at this and subsequent time points, asdescribed in Example 14, and presented as the percentage of time zero.No decrease in luciferase activity (BTL) was seen during the 10 hrtime-course. The 2 hr half-life EGFP construct (BTG2) showed a moderateresponse to the doxycycline-induced block in transcription and a fasterresponse was seen with the 1 hr half-life EGFP (BTG1). The constructcontaining the nonamers (BTG1N4), however, showed by far the fastestresponse to this block in transcription.

FIG. 8 is a graphical representation showing the data used for FIG. 7displayed on a linear scale. The doxycycline-induced block intranscription is detectable as a 50% block in reporter levels afterapproximately 6.5 hrs with BTG1. However, this is reduced to less than 3hrs by inclusion of the nonamers (BTG1N4).

FIG. 9 is a graphical representation showing the effect of differentnumbers (1, 2 or 4) of nonamer RNA destabilising elements. A time-coursewas performed as described in FIG. 7, except with time zero defined as 4hrs after addition of doxycycline to eliminate the effect of the delayin the action of this drug. The presence of a single nonamer (BTG1N1)was sufficient to increase the “effective rate of decay,” whereasprogressively stronger effects were seen with 2 nonamers (BTG1N2) and 4nonamers (BTG1N4). The latter construct showed an “effective half-life”of ˜1 hr 20 mins, which is little more than the 1 hr half-life of theprotein alone.

FIG. 10 is a graphical representation showing changing reporter levelsover time in the absence of a transcriptional block A time-course wasperformed as described in FIG. 7. However, the data presented representsamples not treated with doxycycline and measured at 24 hrs aftertransfection (start) or 34 hrs after transfection (finish). Consistentexpression levels were seen only with BTG1N4.

FIG. 11 is a graphical representation showing changes in reporter levelsover time in the absence of a transcriptional block A time-course wasperformed as described in FIG. 7. BTG1fos contains the c-fos ARE. Thesedata demonstrate that different types of mRNA destabilising elements canbe used to achieve the same effect.

FIG. 12 is a graphical representation showing that RNA destabilisingelements are useful in determining expression when a Luciferase reporterprotein is used. A further enhancement would be expected using aluciferase reporter protein with protein-destabilising elements. Atime-course was performed as described in FIG. 7, using twoluciferase-expressing constructs. BTL contains the standard Fireflyluciferase-coding region and 3′-UTR (derived from pGL3-Basic; Promega),whereas BTLN6 contains 6 copies of the nonamer UUAUUUAUU [SEQ ID NO:1]in the 3′-UTR.

FIG. 13 is a graphical representation showing reporter levels over timeusing DsRed destabilised by RNA destabilising elements andprotein-destabilising elements. A time course was performed as describedin FIG. 7 and Example 14. The constructs used were DsRed2 (BTR),DsRed-MODC (BTR1) and DsRed-MODC containing 4 UUAUUUAUU [SEQ ID NO:1]nonamers in the 3′-UTR (BTR1N4). After blocking transcription withdoxycycline, red fluorescence continues to increase with all constructs.This is substantially reduced by the protein-destabilising element andfurther reduced by the mRNA destabilising element.

FIG. 14 is a graphical representation showing a time-course wasperformed as described in FIG. 7. All of the mRNA destabilising elementstested were very effective at increasing the rate of decay compared tocontrols (BTG1). These data show that the c-myc ARE is an effectivedestabilising element (BTG1myc) and that a modest increase indestabilising activity can be obtained by combining the myc ARE with 4nonamers (BTG1N4myc). Six nonamers (BTG1N6) also appeared to destabilisesomewhat more than 4 nonamers (BTG1N4).

FIG. 15 is a graphical representation showing a time-course similar tothat described in FIG. 7. Five micrograms of each plasmid wastransfected into Tet-Off HeLa cells (Clontech) using Lipofectamine 2000(Gibco BRL). Six to eight hours later, the contents of each flask(transfection) was split into multiple dishes. Twenty four hrs aftertransfection, a drug (doxycycline) known to inhibit transcription fromthe TRE promoter contained in each vector was added to a finalconcentration of one microgram per mL and the cells were harvested atthe time-points shown. Reporter levels (luminescence) were measuredusing the Dual Luciferase Assay kit (Promega) and a luminometer (Wallac)and expressed as a % of time zero. Each plasmid was constructed usingthe pGL3-Basic vector backbone (initial B in plasmid name), with the TREpromoter (T in plasmid name) placed upstream of the Renilla luciferasecoding sequence (Rn in plasmid name). Thus, BTRn contains the standardRenilla luciferase reporter. In BTRn1, the mutant MODCprotein-destabilising sequence (identical to that in the d1EGFP vector(Clontech) was fused, in frame, to the 3′ end of the Renilla luciferasesequence and is denoted by the number 1 immediately after the reporter(Rn) symbol. In BTRnN4, 4 copies of the mRNA destabilising nonamerTTATTTATT (denoted by N4 in the plasmid name) were placed into the3′-UTR-encoding region. In BTRn1N4, both the protein (1)- and mRNA(N4)-destabilising sequences were incorporated. The standard Renillaluciferase reporter decayed very slowly, reaching 50% of initial valuesafter 18 hrs. Modified reporter vectors incorporating either theprotein-destabilising element (BTRn1) or the mRNA-destabilising element(BTRnN4) decayed more rapidly, reaching 50% values at 9-11 hrs. However,the vector containing both protein- and mRNA-destabilising elements,showed, by far, the most rapid response and reached 50% in about 3.5hrs.

FIG. 16 is a graphical representation showing a time-course similar tothat described in FIG. 15, except using plasmids containing the Fireflyluciferase reporter (L in plasmid name). As seen with Renillaluciferase, the standard Firefly luciferase (BTL) decays slowly, taking18 hrs to reach 50%. Modified reporter vectors incorporating either theprotein-destabilising element (BTL1) or the mRNA-destabilising element(BTLN4) decayed more rapidly. However, the vector containing bothprotein- and mRNA-destabilising elements, showed, by far, the most rapidresponse and reached 50% in about 3.5 hrs.

FIG. 17 is a graphical representation showing a time-course similar tothat described in FIG. 15, except using plasmids containing the enhancedgreen fluorescent protein (EGFP) reporter (G in plasmid name). Reporterlevels were measured by flow cytometry and analysed using FlowJosoftware. Briefly, the percentage of positive cells was determined attime zero and used to assign a “percentile” corresponding to the medianof the positive cells. The fluorescence of that percentile was thenmeasured at all time points and expressed as a percentage of the timezero value. As seen with the luciferase vectors, the standard EGFP (BTG)decays slowly, taking ˜20 hrs to reach 50%. Using Clontech'sdestabilised, d2EGFP reporter, with a reported protein half-life of 2hrs (BTG2), the time to 50% was reduced to ˜10 hrs and this was furtherreduced to ˜6.3 hrs by substituting in the strongerprotein-destabilising motif from d1EGFP (BTG1). However, the vectorfurther containing the mRNA-destabilising element (BTG1N4), showed, byfar, the most rapid response and reached 50% in ˜3.3 hrs.

FIG. 18 is a graphical representation showing a time-course similar tothat described in FIG. 15, except using plasmids containing the enhancedyellow fluorescent protein (EYFP) reporter (Y in plasmid name). As seenwith other standard reporter vectors, the standard EYFP (BTY) decaysslowly, taking >24 hrs to reach 50%. Using Clontech's destabilised,d2EYFP reporter, with a reported protein half-life of 2 hrs, the time to50% was reduced to ˜20.5 hrs (BTY2) and this was further reduced to ˜12hrs by substituting in the stronger protein-destabilising motif fromd1EGFP (BTY1). However, the vector further containing themRNA-destabilising element (BTY1N4) showed, by far, the most rapidresponse and reached 50% in ˜4 hrs.

FIG. 19 is a graphical representation showing a time-course similar tothat described in FIG. 15, except using plasmids containing the enhancedcyan fluorescent protein (ECFP) reporter (C in plasmid name). As seenwith other standard reporter vectors, the standard ECFP (BTC) decaysslowly, taking >24 hrs to reach 50%. Using Clontech's destabilised,d2ECFP reporter, with a reported protein half-life of 2 hrs, the time to50% was reduced to ˜16.5 hrs (BTC2) and this was further reduced to ˜12hrs by substituting in the stronger protein-destabilising motif fromd1EGFP (BTC1). However, the vector further containing themRNA-destabilising element (BTC1N4) showed, by far, the most rapidresponse and reached 50% in ˜4.3 hrs.

FIG. 20 is a graphical representation showing a time-course similar tothat described in FIG. 15, except using plasmids containing the HcRedred fluorescent protein (Clontech) reporter (H in plasmid name). As seenwith other standard reporter vectors, the standard HcRed (BTH) decaysslowly, taking >24 hrs to reach 50%. Fusing this reporter gene to theMODC fragment from d2EGFP reduced this time to ˜22.5 hrs (BTH2) and thiswas further reduced to ˜17 hrs by substituting in the strongerprotein-destabilising motif from d1EGFP (BTH1). However, the vectorfurther containing the mRNA-destabilising element (BTH1N4) showed, byfar, the most rapid response and reached 50% in ˜7.3 hrs (4-7 hrs inrepeat experiments).

FIG. 21 is a graphical representation showing a time-course similar tothat described in FIG. 15, except using plasmids containing thebeta-galactosidase reporter (B in plasmid name) from pSV-betagalactosidase (Promega). The standard beta-galactosidase reporter (BTB)showed little, if any decay in activity over 32 hrs. Fusing thisreporter gene to the MODC fragment from d1EGFP (BTB1) caused a slightincrease in decay rate but a faster decay was seen with the vectorsfurther containing the mRNA-destabilising element (BTB1N4) or containingthe mRNA-destabilising element alone (BTBN4). These latter 2 vectorswere further modified (BTuB1N4 and BTuBN4 respectively) to incorporate aubiquitin sequence (u in reporter name), at the 5′ end of theprotein-coding sequence, such that upon cleavage of the ubiquitin, theremaining (modified) beta galactosidase protein contains an N-terminalamino acid sequence beginning with arginine and shown to destabiliseproteins via the N-end rule. These ubiquitin-fusion vectors showed morerapid decay but the fastest decay was achieved with BTmuB1N4, whichcontains a mutant (non-cleavable) ubiquitin sequence at the 5′ codingsequence (mu in reporter name), the MODC fragment at the 3′ codingsequence and the four nonamers in the 3′-UTR sequence.

FIG. 22 is a graphical representation showing a time-course similar tothat described in FIG. 15. BTY1N4 represents the EYFP reporter, withboth protein- and mRNA-destabilising elements as described in FIG. 18.BTpuroY1N4 was constructed by inserting the puromycin coding sequence,in frame, at the 5′ end of the coding sequence in BTY1N4, such that thereporter protein produced is a fusion of the puromycin-resistanceprotein, EYFP and the MODC destabilising sequence. As seen in FIG. 18,reporter levels from BTY1N4 decay rapidly after doxycycline (drug). Asimilar rate of decay was seen with the puromycin-fusion reporter,either when expressed transiently (BTpuroY1N4) or stably (BTpuroY1N4stable cell line). The fact that we were able to select a stable cellline in puromycin shows that the puromycin resistance gene is active inthis fusion protein and the detectable levels of fluorescence show thatthe EYFP component maintains fluorescent activity. The decay curvesdemonstrate that rapid decay of our destabilised reporters isreproducible in stably transfected cells and is not compromised with thefusion protein. Similarly, the neomycin-EYFP-MODC fusion protein(BTneoY1N4) also conferred antibiotic resistance (not shown) andexpressed detectable levels of fluorescence that decayed rapidly afterdrug. FIG. 22B shows a similar but separate experiment utilising thesame BTY1N4 and BTpuroY1N4 constructs. The wild-type ubiquitin sequence,followed by an arginine was cloned in frame and upstream of the codingsequence in these vectors to create BTuY1N4 and BTupuroY1N4respectively. Upon translation of these reporters, the ubiquitinpolypeptide is cleaved, to create a reporter protein with an N-terminalarginine and associated leader sequence that directs decay via the N-endrule. In particular, BTuY1N4 decayed extremely fast, reaching 50% ofinitial values after only ˜1.7 hrs. This demonstrates that enhanceddecay can be achieved by incorporating 2 different protein degradationsignals.

FIG. 23 is a graphical representation showing a time-course followingactivation of transcription via addition of a drug. In this experiment,the drug was PMA (50 ng/mL) and the vectors contained the TRE promoterfollowed by the firefly luciferase coding sequence, either without (DUT)or with (BTL1N4) the protein- and mRNA-destabilising elements. Twoseparate experiments were performed (FIGS. 23A and 23B) and both show a4-5 fold increase in levels of the destabilised reporter (BTL1N4)following PMA. In contrast, the standard, stable reporter shows littledetectable change after PMA. These data show that a moderate increase intranscription is easily detectable with BTL1N4 but is virtuallyundetectable with the standard reporter (BTL).

FIG. 24 is a graphical representation showing a time-course followingactivation of transcription via addition of a drug. As in FIG. 23, thedrug was PMA (50 ng/mL). However, the vectors in this series containedthe Renilla luciferase coding sequence, either without (BNRn) or with(BNRn1N4) the protein- and mRNA-destabilising elements. Moreover, thepromoter was comprised of 4 copies of the NFκB binding sequence (N invector name) in place of the TRE promoter. Compared to the TRE, NF-κB ismore strongly activated by PMA. Two separate experiments were performed(FIGS. 24A and 24B) and both show an ˜8-10 fold increase in levels ofthe destabilised reporter (BNRn1N4) following PMA. In contrast, thestandard, stable reporter (BNRn) shows only a ˜2 fold increase afterPMA. These data, together with the decay data (e.g. FIGS. 15-22) confirmthat both activation and inhibition of transcription are more easilydetected and accurately quantified using the destabilised reportervectors, compared to standard reporter vectors.

FIG. 25 is a graphical representation of an experiment designed to mimica high-throughput drug-screening assay that utilises either the mRNA-and protein-destabilised (FIG. 25A) or standard (FIG. 25B) Renillaluciferase reporters. Cells were transfected with the indicated plasmidsas described in FIG. 15, except that 6-8 hrs after transfection, thecells were trypsinised, counted and then seeded into the wells of a96-well plate at a density of ˜20,000 cells/well. At 24, 26 and 28 hrspost-transfection, PMA (or carrier control; ethanol) was added to 2wells to create duplicate samples representing 2, 4 and 6 hrs drugtreatment plus controls. At 30 hrs, the media was removed, the cellslysed within their wells using Passive Lysis Buffer (Promega), andreporter activity quantified as described in FIG. 15. The raw data(without indication of the drug-treated samples) were transferred toanother scientist, who plotted the data and attempted to identify thesamples containing active drug. This proved very easy with thedestabilised vector (BNRn1N4), even for the shortest drug treatment.With the standard vector (BNRn), however, only the longest drugtreatments could be identified and these showed only a modest 50%increase, compared to 600-700% with the destabilised vector. Theidentities of the drug treated samples were cross-checked and areindicated by symbols in FIG. 25A (BNRn1N4) and FIG. 25B (BNRn).

FIG. 26 is a graphical representation of an experiment that isessentially identical to that shown in FIG. 25, except that the reporterwas firefly luciferase and only two different drug treatments (2 hr and4 hr) were performed. As seen with Renilla luciferase, the performanceof firefly luciferase was also substantially improved by destabilisingthe mRNA and protein (BNL1N4; FIG. 26A) as compared to the standardvector (BNL; FIG. 26B). This is evidenced by the faster and morepronounced change in reporter levels following drug treatment.

FIG. 27 is a graphical representation of an experiment that isessentially identical to that shown in FIG. 25, except utilising the TREas the promoter and doxycycline as the drug so as to mimic a screen fordrugs that inhibit, rather than activate a pathway leading totranscription. Although the inhibition of reporter transcription (thedesired effect) was presumably identical with both vectors, thepercentage change in reporter levels (the measurable effect) was greatlyenhanced with the mRNA- and protein-destabilised Renilla luciferase(BTRn1N4; FIG. 27A) as compared to the standard Renilla vector (BTRn;FIG. 27B).

FIG. 28 is a graphical representation of an experiment similar to thatdescribed in FIG. 7, except using dual-colour vectors based on theexample shown in FIG. 4B. A single TRE promoter drives transcription ofdestabilised HcRed in one direction (BTH1N4) and destabilised EGFP inthe other. (BTG1N4). The 5′-UTR of the EGFP transcript was altered ineach construct to contain a synthetic (artificial) UTR, the Hsp70 5′UTR,the beta globin 5′-UTR or the 5′-UTR from standard Promega andInvitrogen reporter vectors. In puro-GFP, the actual EGFP-coding region(from BTG1N4) was fused with the coding region from the puromycinresistance gene to create BTpuroG1N4. FIG. 28A shows the ratio of greenfluorescence to red fluorescence (in the absence of drug), expressedrelative to the ratio with Promega 5′-UTR construct. The poor level oftranslation with the synthetic 5 ′-UTR, as well as the translationalenhancer activities of Hsp70 and beta-globin 5′-UTRs are clearlyevident. Interestingly, the puro-green construct appears to express ateven higher levels. FIG. 28B shows that following a block intranscription, EGFP fluorescence decays at a similar rate with allconstructs. This demonstrates that the different “steady state”expression levels seen in FIG. 28A are not caused by an effect of the5′-UTR on mRNA (or protein) stability.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions and Abbreviations

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “5′-UTR” is meant the 5′ (upstream) untranslated region of a gene.Also used to refer to the DNA region encoding the 5′-UTR of the mRNA.

By “3′-UTR” is meant the region of a polynucleotide downstream of thetermination codon of a protein-encoding region of that polynucleotide,which is not translated to produce protein.

By “about” is meant a quantity, level, value, dimension, size, or amountthat varies by as much as 30%, preferably by as much as 20%, and morepreferably by as much as 10% to a reference quantity, level, value,dimension, size, or amount.

By “ARE” is meant an AU-rich element in mRNA i.e., a sequence thatcontains a high proportion of adenine and uracil nucleotides. Also usedto refer to the DNA region encoding such a mRNA element.

By “biologically active fragment” is meant a fragment of a full-lengthreference polynucleotide or polypeptide which fragment retains theactivity of the reference polynucleotide or polypeptide, respectively.

By “CAT” is meant chloramphenicol acetyltransferase.

As used herein, the term “cis-acting sequence,” “cis-acting regulatoryelement,” “cis-regulatory region,” “regulatory region” and the likeshall be taken to mean any sequence of nucleotides, which whenpositioned appropriately relative to a transcriptional control elementor to a transcribable sequence, is capable of modulating, at least inpart, the activity of the transcriptional control element or theexpression of the transcribable sequence. Those skilled in the art willbe aware that a cis-acting regulatory element may be capable ofactivating, silencing, enhancing, repressing or otherwise altering thelevel of expression and/or cell-type-specificity and/or developmentalspecificity of a gene sequence at the transcriptional orpost-transcriptional level. In some embodiments of the presentinvention, the cis-acting regulatory element is an activator sequencethat enhances or stimulates the activity of a transcriptional controlelement or the expression of a transcribable sequence. In otherembodiments, the cis-acting regulatory element modulates mRNA stability,mRNA processing and export or translation.

By “d1EGFP” is meant a variant of EGFP that is fused to a mutated PESTsequence and consequently has a half-life of only about 1 hour.Similarly, d1ECFP and d1EYFP are also available. A destabilised variantof DsRed could be made in the same way. Henceforth referred to asd1DsRed.

By “d2EGFP” is meant a mutant form of EGFP variants that is fused to aPEST sequence and consequently has a half-life of only 2 hours.Similarly, d2ECFP (cyan) and d2EYFP (yellow) are also available. Adestabilised variant of DsRed could possibly be made in the same way.Henceforth referred to as d2DsRed.

By “dEGFP” is meant a general term for all destabilised variants of EGFP(including all colours) formed. (Li et al).

By “derivative” is meant a polynucleotide or polypeptide that has beenderived from a reference polynucleotide or polypeptide, respectively,for example by conjugation or complexing with other chemical moieties orby post-transcriptional or post-translational modification techniques aswould be understood in the art.

By “DNA” is meant deoxyribonucleic acid.

By “DsRed” is meant the red fluorescent protein isolated from theIndoPacific sea anemone relative Discosoma species.

By “ECFP” is meant the mutant form of EGFP with alteredexcitation/emission spectra that fluoresces cyan coloured light.

By “EGF” is meant epidermal growth factor

By “EGFP” is meant the enhanced green fluorescent protein. A mutant formof GFP with enhanced fluorescence. (Cormack et al).

By “ErbB2” is meant the second member of the epidermal growth factorreceptor family. Also known as HER-2.

By “exon” is meant the sequences of a RNA primary transcript that arepart of a messenger RNA molecule, or the DNA that encodes suchsequences. In the primary transcript neighbouring exons are separated byintrons.

By “expression vector” is meant a vector that permits the expression ofa polynucleotide inside a cell. Expression of a polynucleotide includestranscriptional and/or post-transcriptional events

By “EYFP” is meant a mutant form of EGFP with alteredexcitation/emission spectra that fluoresces yellow coloured light.

By “firefly luciferase” is meant the enzyme derived from the luc gene ofthe firefly, which catalyses a reaction using D-luciferin and ATP in thepresence of oxygen and Mg⁺⁺ resulting in light emission.

By “flow cytometry” is meant a method, in which live or fixed cellsuspensions are applied to a flow cytometer that individually measuresan activity or property of a detectable label associated with the cellsof the suspension. Labelling of cells can occur, for example, viafluorescent compounds or by antibodies covalently attached to a specificfluorescent compound. Several different excitation/emission wavelengthscan be tested simultaneously to measure different types of fluorescence.Sub-populations of cells with desired characteristics (fluorescence,cell size) can be gated such that further statistical analyses applyonly to the gated cells. Flow cytometers equipped with a cell sortingoption can physically separate cells with the desired fluorescence andretrieve those (live) cells in a tube separate from the remainder of theinitial cell population. Also referred to as FACS (fluorescenceactivated cell sorting.

The term “gene” as used herein refers to any and all discrete codingregions of a host genome, or regions that code for a functional RNA only(e.g., tRNA, rRNA, regulatory RNAs such as ribozymes etc) as well asassociated non-coding regions and optionally regulatory regions. Incertain embodiments, the term “gene” includes within its scope the openreading frame encoding specific polypeptides, introns, and adjacent 5′and 3′ non-coding nucleotide sequences involved in the regulation ofexpression. In this regard, the gene may further comprise controlsignals such as promoters, enhancers, termination and/or polyadenylationsignals that are naturally associated with a given gene, or heterologouscontrol signals. The gene sequences may be cDNA or genomic DNA or afragment thereof. The gene may be introduced into an appropriate vectorfor extrachromosomal maintenance or for integration into the host.

By “GFP” is meant a fluorescent protein (Tsien et al), which isisolatable from the jellyfish Aequoria victoria, and which can be usedas a reporter protein. DNA constructs encoding GFP can be expressed inmammalian cells and cause the cells to fluoresce green light whenexcited with specific wavelengths. The term “GFP” is used herein torefer to all homologues and analogues, including colour variants andfluorescent proteins derived from organisms other than Aequoria victoria(e.g., DSRed and HcRed, Clonetech; hrGFP, Stratagene).

By “half-life” is meant the time taken for half of the activity, amountor number of molecules to be eliminated. Thus, the “mRNA half-life” isthe time taken for half of the existing mRNA molecules to decay. mRNAhalf-life can be measured by blocking transcription (e.g. withActinomycin D) and measuring the rate of decay of the mRNA in theabsence of any new mRNA being formed. Alternatively, the intracellularhalf-life of a polypeptide refers to the time taken for half of theactivity, amount or number of polypeptide molecules in a cell orpopulation of cells to decay. Polypeptide half-life can be measured byblocking translation (e.g. with cyclohexamide) and measuring the rate ofdecay of the polypeptide (or its functional activity) in the absence ofany new polypeptide being formed. However, the use of polypeptide levelsor activities as a measure of gene expression at earlier stages such astranscription, suffers from long time delays between the actual effect(altered transcription) and the measurable effect (altered proteinlevels). A major cause of this delay is the relatively slow decay ofboth the mRNA and the protein. The effects of this delay include: minoror transient changes in transcription are difficult to detect; kineticassays are highly inaccurate; and assays require long incubation times.Accordingly, the true measure of the time delay between alteredtranscription and altered protein levels would be the rate of decay ofthe polypeptide after a block in transcription, which would incorporatethe combined effects of protein stability and mRNA stability. Thismeasurement is referred to herein as the “effective half-life” of apolypeptide.

By “intron” is meant a non-coding sequence within a gene, or its primarytranscript, that is removed from the primary transcript and is notpresent in a corresponding messenger RNA molecule.

By “luciferase” is meant any reporter enzyme that catalyses a reaction,which leads to light emission. Exogenous substrates are added and thereaction is quantified using a luminometer. The substrate requirementsfor firefly and Renilla luciferases are different, allowing the two tobe distinguished in the Dual Luciferase Assay (Promega, Madison, Wis.,USA).

By “MAPK” is meant a mitogen activated protein kinase. Includes severaldifferent kinases involved in intracellular signal transduction pathwaysthat lead to growth or apoptosis (cell death). The term “MAPK” issometimes used in reference to two specific MAPKs, Erk1 and Erk2(extracellular regulated kinases 1 and 2).

By “mCMV” is meant a minimal CMV promoter. In some embodiments, aminimal mCMV promoter does not activate transcription on its own but canbe linked to a TRE to provide tetracycline (and doxycycline)-dependenttranscription or linked to other enhancer elements to providetranscription that is dependent on the activity of that enhancer.

By “MCS” is meant a multiple cloning site, which is a region of anucleic acid molecule comprising a plurality of sites cleavable bydifferent enzymes or chemicals for inserting polynucleotides into thenucleic acid molecule. Typically, a MCS refers to a region of a DNAvector that contains unique restriction enzyme recognition sites intowhich a DNA fragment can be inserted. The term “MCS” as used herein,also includes any other site that assists the insertion of DNA fragmentsinto the vector. For example, a T overhang (Promega, Madison, Wis.,USA), which allows direct insertion of fragments generated by polymerasechain reaction (PCR).

By “mRNA” is meant messenger RNA, which is a “transcript” produced in acell using DNA as a template, which itself encodes a protein. mRNA istypically comprised of a 5′-UTR, a protein encoding (i.e., coding)region and a 3′-UTR. mRNA has a limited half-life in cells, which isdetermined, in part, by stability elements, particularly within the3′-UTR but also in the 5′-UTR and protein encoding region.

By “MODC” is meant mouse ornithine decarboxylase or a portion, variantor derivative thereof containing a PEST sequence.

By “modulating” is meant increasing or decreasing, either directly orindirectly, the stability or activity of a molecule of interest.

By “operably connected” or “operably linked” and the like is meant alinkage of polynucleotide elements in a functional relationship. Anucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the coding sequence. Operably linked meansthat the nucleic acid sequences being linked are typically contiguousand, where necessary to join two protein coding regions, contiguous andin reading frame. A coding sequence is “operably linked to” anothercoding sequence when RNA polymerase will transcribe the two codingsequences into a single mRNA, which is then translated into a singlepolypeptide having amino acids derived from both coding sequences. Thecoding sequences need not be contiguous to one another so long as theexpressed sequences are ultimately processed to produce the desiredprotein. “Operably connecting” a promoter to a transcribablepolynucleotide is meant placing the transcribable polynucleotide (e.g.,protein encoding polynucleotide or other transcript) under theregulatory control of a promoter, which then controls the transcriptionand optionally translation of that polynucleotide. In the constructionof heterologous promoter/structural gene combinations, it is generallypreferred to position a promoter or variant thereof at a distance fromthe transcription start site of the transcribable polynucleotide, whichis approximately the same as the distance between that promoter and thegene it controls in its natural setting; i.e.: the gene from which thepromoter is derived. As is known in the art, some variation in thisdistance can be accommodated without loss of function. Similarly, thepreferred positioning of a regulatory sequence element (e.g., anoperator, enhancer etc) with respect to a transcribable polynucleotideto be placed under its control is defined by the positioning of theelement in its natural setting; i.e. the genes from which it is derived.

The term “pA” as used herein refers to a polyadenylation site, which isa DNA sequence that serves as the site to cleave and add to the immaturemRNA, a polyA tail. Various pA sequences from SV40 virus genes or the βgalactosidase gene or other sources, including synthetic polyadenylationsites can be used in expression vectors for this purpose.

The term “PEST” refers to an amino acid sequence that is enriched withthe amino acids proline (P), glutamic acid (E), serine (S) and threonine(M). Proteins containing PEST sequences have shortened half-lives.

By “plasmid” is meant a circular DNA vector. Plasmids contain an originof replication that allows many copies of the plasmid to be produced ina bacterial (or sometimes eukaryotic) cell without integration of theplasmid into the host cell DNA.

By “PMA4” is meant phorbol myristoloic acid.

By “polynucleotide” or “nucleic acid” is meant linear sequences ofnucleotides, including DNA or RNA, which may be double-stranded orsingle-stranded.

By “polypeptide,” “peptide” or “protein” is meant a polymer of aminoacids joined by peptide bonds in a specific sequence.

By “promoter” is meant a region of DNA, generally upstream (5′) of acoding region, which controls at least in part the initiation and levelof transcription. Reference herein to a “promoter” is to be taken in itsbroadest context and includes the transcriptional regulatory sequencesof a classical genomic gene, including a TATA box and CCAAT boxsequences, as well as additional regulatory elements (i.e., activatingsequences, enhancers and silencers) that alter gene expression inresponse to developmental and/or environmental stimuli, or in atissue-specific or cell-type-specific manner. A promoter is usually, butnot necessarily, positioned upstream or 5′, of a structural gene, theexpression of which it regulates. Furthermore, the regulatory elementscomprising a promoter are usually positioned within 2 kb of the startsite of transcription of the gene. Promoters according to the inventionmay contain additional specific regulatory elements, located more distalto the start site to further enhance expression in a cell, and/or toalter the timing or inducibility of expression of a structural gene towhich it is operably connected. The term “promoter” also includes withinits scope inducible, repressible and constitutive promoters as well asminimal promoters. Minimal promoters typically refer to minimalexpression control elements that are capable of initiating transcriptionof a selected DNA sequence to which they are operably linked. In someexamples, a minimal promoter is not capable of initiating transcriptionin the absence of additional regulatory elements (e.g., enhancers orother cis-acting regulatory elements) above basal levels. A minimalpromoter frequently consists of a TATA box or TATA-like box. Numerousminimal promoter sequences are known in the literature. For example,minimal promoters may be selected from a wide variety of knownsequences, including promoter regions from fos, CMV, SV40 and IL-2,among many others. Illustrative examples are provided which use aminimal CMV promoter or a minimal IL2 gene promoter (−72 to +45 withrespect to the start site; Siebenlist, 1986).

By “Renilla luciferase” is meant a polypeptide, which is derivable fromsea pansy (Renilla reniformis), and which utilizes oxygen andcoelenterate luciferin (coelenterazine) to generate light emission.

By “reporter vector” is meant an expression vector containing a“reporter gene” that encodes a polypeptide (or mRNA) that can be easilyassayed. Typically, the reporter gene is linked to regulatory sequences,the function or activity of which, is being tested.

By “reporter” is meant a molecule, typically a protein or polypeptide,which is encoded by a reporter gene and measured in a reporter assay.Current systems generally utilize an enzymatic reporter and measurereporter activity.

By “RNA” is meant ribonucleic acid.

By “rtTA” is meant reverse tTA (see below), which binds the TRE andactivates transcription only in the presence of tetracycline ordoxycycline.

By “SEAP” is meant secreted alkaline phosphatase reporter gene.

By “SKBR3” is meant the human breast cancer cell line that overexpressesErbB2.

By “stringent conditions” is meant temperature and ionic conditionsunder which only nucleotide sequences having a high frequency ofcomplementary bases will hybridise. The stringency required isnucleotide sequence dependent and depends upon the various componentspresent during hybridisation and subsequent washes, and the time allowedfor these processes. Generally, in order to maximise the hybridisationrate, non-stringent hybridisation conditions are selected; about 20 to25° C. lower than the thermal melting point (T_(m)). The T_(m) is thetemperature at which 50% of specific target sequence hybridises to aperfectly complementary probe in solution at a defined ionic strengthand pH. Generally, in order to require at least about 85% nucleotidecomplementarity of hybridised sequences, highly stringent washingconditions are selected to be about 5 to 15° C. lower than the T_(m). Inorder to require at least about 70% nucleotide complementarity ofhybridised sequences, moderately stringent washing conditions areselected to be about 15 to 30° C. lower than the T_(m). Highlypermissive (low stringency) washing conditions may be as low as 50° C.below the T_(m) allowing a high level of mis-matching between hybridisedsequences. Those skilled in the art will recognise that other physicaland chemical parameters in the hybridisation and wash stages can also bealtered to affect the outcome of a detectable hybridisation signal froma specific level of homology between target and probe sequences.

The term “SV40/CMV/RSV” is used herein to refer to promoter elementsderived from simian virus, cytomegalovirus and Rous sarcoma virusrespectively. Generally, these promoters are thought to beconstitutively active in mammalian cells.

By “TetO” is meant the Tet operator DNA sequence derived from the E.coli tetracycline-resistance operon.

By “Tet-Off Cell Lines” is meant cell lines stably expressing rtTA suchthat tetracycline or doxycycline will shut off transcription from TREpromoters.

By “Tet-On Cell Lines” is meant cell lines stably expressing rtTA suchthat tetracycline or doxycycline will turn on transcription from TREpromoters.

By “transcription” is meant the process of synthesizing a RNA moleculecomplementary to the DNA template.

By “transfection” is meant the process during which a plasmid or DNAfragment is inserted into a eukaryotic cell. Typically, 2-50% of cellstake up the plasmid and express the protein product for ˜3 days withoutincorporating the plasmid DNA into the cell's chromosomes (=transienttransfection). A small proportion of these cells will eventuallyincorporate the plasmid DNA into their genome and permanently expressthe protein product (=stable transfection).

As used herein the term “transgenic” refers to a genetically modifiedanimal in which the endogenous genome is supplemented or modified by therandom or site-directed integration of a foreign gene or sequence.

The “transgenic animals” of the invention are suitably produced byexperimental manipulation of the genome of the germline of the animal.These genetically engineered animals may be produced by several methodsincluding the introduction of a “transgene” comprising nucleic acid(usually DNA) into an embryonal target cell or integration into achromosome of the somatic and/or germ line cells of an animal by way ofhuman intervention. A transgenic animal is an animal whose genome hasbeen altered by the introduction of a transgene.

By “translation” is meant the process whereby a mRNA molecule is used asa template for protein synthesis.

By “TRE” is meant any tetracycline responsive element (Gossen et al),generally combined with a minimal promoter such that transcriptionoccurs only via the binding of exogenous factors (e.g., tTA or rtTA) tothe TRE. Preferred embodiments of this invention utilize a TRE comprisedof 7 repeats of the tetO sequence linked to a minimal CMV promoter(mCMV) (Clontech Laboratories Inc., Palo Alto, Calif., USA).

By “tTA” is meant tetracycline-controlled transactivator, which iscomprised of the Tet repressor protein (TetR) and the VP16 activationdomain, such that it binds the TRE and activates transcription, only inthe absence of tetracycline or doxycycline.

By “TS” is meant thromboxane synthase promoter.

By “variant” is meant a polynucleotide or polypeptide displayingsubstantial sequence identity with a reference polynucleotide orpolypeptide, respectively. Variant polynucleotides also includepolynucleotides that hybridise with a reference sequence under stringentconditions. These terms also encompasses polynucleotides which differfrom a reference polynucleotide by the addition, deletion orsubstitution of at least one nucleotide. In this regard, it is wellunderstood in the art that certain alterations inclusive of mutations,additions, deletions and substitutions can be made to a referencepolynucleotide whereby the altered polynucleotide retains the biologicalfunction or activity of the reference polynucleotide. The terms“polynucleotide variant” and “variant” also include naturally occurringallelic variants. With regard to variant polypeptides, it is wellunderstood in the art for example that some amino acids may be changedto others with broadly similar properties without changing the nature ofthe activity of the polypeptide (conservative substitutions).

By “vector” is meant a vehicle for inserting a foreign DNA sequence intoa host cell and/or amplifying the DNA sequence in cells that supportreplication of the vector. Most commonly a plasmid but can also be aphagemid, bacteriophage, adenovirus or retrovirus.

By “vEGFP,” “EGFP I” or “variant of EGFP” is meant different colourvariants and/or different half-life variants of EGFP.

By “vGFP” is meant all variants of GFP; including homologues andanalogues such as DsRed, also EGFP variants or destabilised GFPvariants.

2. Constructs and Methods of the Present Invention

The present invention provides inter alia expression constructs whichmodulate the stability of transcripts and consequently, the amount oractivity of polypeptide produced by the constructs. Although constructswhich increase the stability of a transcript are clearly encompassed bythe present invention, certain embodiments focus on destabilisingtranscripts. Here transcript stability can be reduced by the addition ofone or more destabilising elements to, or by the removal of one or morestability elements (e.g., a poly A tail) from, a transcribablepolynucleotide. Compared to existing expression constructs, theconstruct of the present invention provides kinetics of proteinexpression with improved temporal correlation to the promoter activity,e.g., by reducing the time lag between decreased promoter activity anddecreased levels of a corresponding expression product or by reducingthe steady state level of the expression product such that increasedpromoter activity results in a larger and/or faster increase in levelsof the expression product relative to that present before the increasein promoter activity.

Accordingly, one aspect of the present invention is directed to aconstruct comprising in operable linkage: a polynucleotide that encodesa polypeptide and a nucleic acid sequence that encodes a RNA elementthat modulates the stability of a transcript encoded by thepolynucleotide.

The term “modulates” in the context of transcript stability refers toincreasing or decreasing the stability of a transcript and optimalamounts of modulation depends upon the particular application. Withoutlimiting the present invention to any one particular theory or mode ofoperation, where the RNA element is a sequence of nucleotides whichdestabilises the transcript, it is envisaged that the element directlyor indirectly targets the transcript for degradation.

As used herein the term “destabilising element” refers to a sequence ofamino acids or nucleotides which reduces the half-life of a protein ortranscript, respectively, inside a cell. Accordingly, a “RNAdestabilising element” comprises a sequence of nucleotides which reducesthe intracellular half-life of a RNA transcript and a“protein-destabilising element” comprises a sequence of amino acidswhich reduces the intracellular half-life of a protein. mRNAdestabilising elements improve the temporal correlation between alteredpromoter activity (or mRNA processing events) and altered cytoplasmicmRNA levels. Protein destabilising elements improve the temporalcorrelation between altered cytoplasmic mRNA levels and altered reporterlevels or activity. The extent of the reduction sought at each leveldepends upon the particular application. In certain embodiments thecombination of RNA destabilisation and protein destabilisationsignificantly improves the temporal correlation between promoteractivity (or mRNA processing) and reporter levels or activity inexpression constructs compared to constructs without destabilisationelements or with only one type of destabilising element. In otherembodiments, the protein destabilising elements improve the temporalcorrelation between altered mRNA stability, processing or translationand altered reporter levels or activity. In relation to increasingtranscript stability, optimum levels of stability will again depend uponthe application.

A “RNA stabilising element” is a sequence of nucleotides which increasesthe intracellular half-life of a RNA molecule which is; suitably, butnot exclusively selected from mRNA, heterogenous nuclear RNA (hnRNA),small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), smallcytoplasmic RNA (scRNA), ribosomal RNA (rRNA), translational control RNA(tcRNA), transfer RNA (tRNA), eRNA, messenger-RNA-interferingcomplementary RNA (micRNA) or interference RNA (iRNA) and mitochondrialRNA (mtRNA). In certain embodiments the RNA molecules are mRNAmolecules.

In the context of reducing the intracellular half-life of a moleculeselected from a RNA transcript or an encoded protein of interest, (a)one or more destabilising elements are typically added and/or (b) one ormore stabilising elements are typically removed in order to confer alevel of enhanced degradation on the molecule, which thereby reduce(s)the intracellular half-life of the molecule to a half-life that issuitably less than about 24, 10 or 5 hours, desirably less than about 3,2 or 1 hour(s), or even less than about 30, 15, 10, 5 or 3 minutes. Thehalf-life of a RNA transcript or an encoded protein of interestadvantageously corresponds to the lowest half-life that provides asteady-state expression level of at least about 10-fold the minimumdetectable level of the transcript or encoded protein.

The intracellular or intracellular-like conditions are typicallyphysiological for the cell type. The temperature of the intracellular orintracellular-like conditions is usually physiological for the celltype. Exemplary temperatures for mammalian cells range generally fromabout 30° C. to about 42° C., and typically from about 35° C. to about37° C.

At a minimum, enhanced ribonucleic or proteolytic degradation of a RNAtranscript or polypeptide, respectively, refers to a level ofribonucleic or proteolytic degradation that is at least about 5, 10, 20,40, 50, 60, 70, 80, 90, 100, 150, 200, 400, 600, 1,000, 2,000, 4,000,6,000, 8,000, 10,000, 12,000% greater than that of the RNA transcript orpolypeptide in the absence of the destabilising element(s) or in thepresence of a stabilising element(s) as the case may be. Assays formeasuring RNA degradation are known to those of skill in the art. Forexample, RNA degradation can be measured using a range of assaysdisclosed for example by Ross, J (1995) or by Liu, J et al. (JBC 2000),which are based on the use of transcriptional inhibitors (Actinomycin D,DRB, cordycepin, alpha-amanitin), pulse labelling (radioactivenucleosides), cell-free decay methods polysomes, cytosol orreticulocytes), or short-term promoter activation (fos promoter, seebelow). Assays for measuring degradation of proteins are also known topersons of skill in the art. For example, proteolytic degradation may bemeasured in vitro using a mammalian cell lysate assay including, but notrestricted to, the reticulocyte lysate assay of Bachmair et al in U.S.patent Ser. No. 5,646,017. Alternatively, proteolytic degradation may bemeasured in vivo using cyclohexamide or pulse-chase protocols as forexample disclosed by Vazhappilly, R and Sucher, N (2002) or by Saito, Tet al. (1998). In certain embodiments, intracellular half-lives ofpolypeptides are determined using blockers of translation (e.g.,cyclohexamide).

The RNA destabilising elements can be derived from any source and inparticular the 3′-UTR or 5′-UTR regions of short-lived mRNAs oftencontain destabilising sequences. As used herein, the term “derived from”shall be taken to indicate that a particular integer or group ofintegers has originated from the species specified, but has notnecessarily been obtained directly from the specified source. Forexample, RNA destabilising sequences may be cloned from short-lived RNAssuch as but not limited to RNAs from the c-fos, c-jun, c-myc, GM-CSF,IL-3, TNF-alpha, IL-2, IL-6, IL-8, IL-10, Urokinase, bcl-2, SGLT1(Na(+)-coupled glucose transporter), Cox-2 (cyclooxygenase 2), IL8,PAI-2 (plasminogen activator inhibitor type 2), beta1-adrenergicreceptor and GAP43 (5′-UTR and 3′-UTR) genes. Alteratively, RNAdestabilising sequences may be selected from AU-rich elements (AREs)and/or U-rich elements (UREs), including but not limited to single,tandem or multiple or overlapping copies of the nonamerUUAUUUA(U/A)(U/A) [SEQ ID NO:2] (where U/A is either an A or a U)(Lagnado et al 1994) and/or the pentamer AUUUA [SEQ ID NO:3] (Xu et al997) and/or the tetramer AUUU [SEQ ID NO:4] (Zubiaga et al. 1995). Theterm “tandem copies” allows for both duplication and/or non-duplicationof one or more of the outer nucleotides. For example, tandem copies ofthe pentamer AUUUA [SEQ ID NO:3], includes sequences such asAUUUAUUUAUUUA [SEQ ID NO:5] as well as AUUUAAUUUAAUUUA [SEQ ID NO:6].RNA destabilising elements have also been described for example fromphosphoenolpyruvate carboxy kinase mRNA (PEPCK), the Drosophila Bicoidgene, the human thioredoxin gene, heat stable antigen gene and soybean10A5 gene.

Iron responsive elements and iron regulatory protein binding sites mayalso be advantageously incorporated into the instant constructs tomodulate RNA stability or translational efficiency experimentally or inresponse to stimuli. Histone RNAs, particularly their 3′-UTRs, areespecially useful for modulating RNA stability in a cell-cycle dependentfashion.

Also contemplated are modifications to or permutations of the elementslisted above. Accordingly, biologically active fragments as well asvariants and derivatives of the destabilising elements referred to aboveare encompassed by the present invention. For example, RNA destabilisingelements may be identified and/or modifications made thereto using acomputational approach and database analysis (Dandekar T et al).

In a related embodiment the present invention contemplates a constructcomprising in operable linkage: a polynucleotide which encodes apolypeptide and a nucleic acid sequence which encodes a stabilising RNAelement that enhances the stability of a transcript encoded bypolynucleotide. In illustrative examples the nucleic acid sequence is,or is derived from, a gene selected from alpha2 globin, alphal globin,beta globin, or growth hormone, which are examples of long-lived mRNAs.As used herein, underscoring or italicising the name of a gene shallindicate the gene, in contrast to its protein product, which isindicated by the name of the gene in the absence of any underscoring oritalicising. For example, “alpha2 globin” shall mean the alpha2 globingene, whereas “alpha2 globin” shall indicate the protein product of the“alpha2 globin” gene.

The ability to destabilise a transcript and to reduce the amount ofprotein produced by a cell will clearly be useful for a wide range ofapplications, including methods for assaying the activity of geneexpression-modulating elements (e.g., transcriptional control elementsand cis-acting regulatory elements) or for identifying elements of thistype or agents that modulate their activity. Thus, another aspect of thepresent invention contemplates a construct comprising in operablelinkage a polynucleotide which encodes a polypeptide and a nucleic acidsequence which encodes a RNA destabilising element that reduces thestability of a transcript encoded by the polynucleotide. In illustrativeexamples, the nucleic acid sequence is, or is derived from, a geneselected from c-fos, c-jun, c-myc, GM-CSF, IL3, TNF-alpha, IL-2, IL-6,IL-8, IL-10, Urokinase, bcl-2, SGLT1 (Na(+)-coupled glucosetransporter), Cox-2 (cyclooxygenase 2), IL-8, PAI-2 (plasminogenactivator inhibitor type 2), beta1-adrenergic receptor or GAP43. Incertain embodiments, the nucleic acid sequence is selected from any oneof SEQ ID NOS 1 to 23, especially from SEQ ID NO:1, 13, 19 or 49, orbiologically active fragments thereof, or variants or derivatives ofthese.

In certain embodiments, the nucleic acid sequence encoding the RNAdestabilising element is linked to a sequence encoding a protein ofinterest, which in turn is linked to a promoter of interest that isoptionally modulatable (i.e., inducible or repressible) such thatexpression is turned on and then turned off. In this application, theRNA destabilising elements typically serve to shorten the period ofexpression of a functional mRNA or protein. This may be applied in vitroor in vivo. For example, a cell cycle-specific promoter could becombined with the RNA destabilising elements to express a protein ofinterest, exclusively in certain stages of the cell cycle. The proteinof interest may be a functional protein or a reporter protein. In thelatter example, reporter levels can be used as an indicator ofcell-cycle stage or cell proliferation. Similarly, the reporter levelsmay be used to give a measure of other cellular events relating to theactivity of the promoter of interest. Such events include, but are notlimited to, apoptosis, immune function, modulation of a signaltransduction pathway, modulation of a regulatory pathway, modulation ofa biosynthetic pathway, toxic response and cell differentiation. Incertain embodiments in which the activity of a cis-acting regulatoryelement (e.g., an enhancer or post-transcriptional control element) isof interest, the reporter levels may also give a measure of cellularevents (as discussed for the promoter of interest) relating to theactivity of that cis-acting regulatory element. By extension, reporterlevels may be used as an indirect measure of action of a compound ortreatment on a cellular event.

One particular application is in the area of determining geneexpression. Specifically, by reducing the time lag between alteredtranscription and altered levels of the resultant polypeptide in a cell,it is possible to more accurately determine promoter or enhanceractivity. In this application a reporter gene, whose expression ismodulated by regulatory elements within the construct, is used todetermine promoter or enhancer activity. Thus, another aspect of thepresent invention contemplates a construct comprising in operablelinkage: a polynucleotide which encodes a reporter polypeptide and anucleic acid sequence which encodes a RNA destabilising element thatreduces the stability of a transcript encoded by the polynucleotide.

In some embodiments the RNA destabilising sequences are incorporatedinto the region encoding the 3′-UTR of the reporter mRNA. Alternativelyor in addition, destabilising elements are incorporated into the 5′-UTRand/or protein coding region, which is suitably not essential to, ordoes not interfere with, the selected activity of the encoded protein.

In a related embodiment the RNA destabilising sequences are used toalter the kinetics of expression of a polypeptide from a gene ofinterest when for example there is a need to accurately monitor, limitor reduce its expression. Typically for this application, RNAdestabilising elements are used in conjunction withprotein-destabilising elements.

The constructs of the present invention have applications in a varietyof gene expression systems where it is desirable to have a brief periodof mRNA or protein expression or to minimise the time lag betweenchanges in promoter activity and the resultant changes in mRNA/proteinlevels.

In certain embodiments, the constructs are designed for use ineukaryotic cell systems. It should be noted however that the RNAdestabilising elements may be used in a wide range of eukaryotic (e.g.,mammals and plants) systems including cells, tissues or whole organismsdefined as yeast, insect, nematode, fish, bird or mammal. For use inplants, different promoters and possibly different reporters and RNAdestabilising elements (e.g., DST sequences) may be used.

In some embodiments, the polynucleotide whose transcript is stabilisedor destabilised by the RNA element encodes a reporter molecule or adestabilised variant thereof. Reporter molecules are well known in theart. Suitably, the reporter molecules are reporter proteins illustrativeexamples of which include, but are not limited to, Luciferase, GreenFluorescent Protein, Red Fluorescent Protein, SEAP, CAT, or biologicallyactive fragments thereof, or variants or derivatives of these.

In other embodiments, the polynucleotide whose transcript is stabilisedor destabilised by the RNA element encodes a protein having at least alight-emitting activity and a selection marker activity. Suitably, thepolynucleotide comprises a chimeric gene, which includes a codingsequence from a gene encoding a light-emitting protein and a codingsequence from a gene encoding a selectable marker protein. Illustrativeexamples of light-emitting proteins include fluorescent proteins (e.g.,Green Fluorescent Protein, Red Fluorescent Protein and their variantsand derivatives) and luminescent proteins (e.g., Luciferases such asRenilla luciferase and Photinus Luciferase and their variants andderivatives). Illustrative examples of selectable marker proteins arepositive selectable marker proteins including, but not limited to,proteins encoded by antibiotic resistance genes (e.g., hygromycinresistance genes, neomycin resistance genes, tetracycline resistancegenes, ampicillin resistance genes, kanamycin resistance genes,phleomycin resistance genes, bleomycin resistance genes, geneticinresistance genes, carbenicillin resistance genes, chloramphenicolresistance genes, puromycin resistance genes, blasticidin-S-deaminasegenes), heavy metal resistance genes, hisD genes, hypoxanthinephosphoribosyl transferase (HPRT) genes and guanine phosphoribosyltransferase (Gpt) genes. In certain embodiments, the light-emittingprotein is selected from Green Fluorescent Protein, Luciferase orbiologically active fragments thereof, or variants or derivatives ofthese and the selectable marker protein is selected from kanamycinkinase, neomycin phosphotransferase, aminoglycoside phosphotransferase,puromycin N-acetyl transferase, puromycin resistance protein orbiologically active fragments thereof, or variants or derivatives.Chimeric genes of this type can be constructed using standardrecombinant or synthetic techniques, as described for example in U.S.Patent Application Publication No. 2002/0150912 and in European PatentApplication No. 1 262 553.

Another aspect of the present invention contemplates the combination ofa protein-destabilising element (e.g., a DNA/RNA sequence encoding anintracellular protein degradation signal or degron which may be selectedfrom a destabilising amino acid at the amino-terminus of a polypeptideof interest, a PEST region or a ubiquitin) and a RNA destabilisingelement (e.g., multiple copies of the nonamer UUAUUUAUU [SEQ ID NO:1]),such that both RNA and protein are destabilised. For example, one suchembodiment incorporates into a construct a PEST sequence immediatelyupstream of the translation stop codon and 4 nonamers located downstreamof the stop codon (suitably 20 nt or more from stop codon). In this way,reporter protein may be destabilised both at the protein level and theRNA (especially mRNA) level.

The destabilised reporter protein may be any suitable protein. Forexample, destabilised GFP proteins are suitable, such as for exampled1EGFP, d1EYFP and d1ECFP comprising the d1 mutant of MODC. Thedestabilised luciferase protein has been described by Leclerc G. et al.The MODC from d1EGFP is also contemplated.

Any method of destabilising a polypeptide of interest is contemplated bythe present invention. For example, a polypeptide of interest can bemodified to include a destabilising amino acid at its amino-terminus sothat the protein so modified is subject to the N-end rule pathway asdisclosed, for example, by Bachmair et al in U.S. patent Ser. No.5,093,242 and by Varshavsky et al. in U.S. patent Ser. No. 5,122,463. Insome embodiments, the destabilising amino acid is selected fromisoleucine and glutamic acid, especially from histidine tyrosine andglutamine, and more especially from aspartic acid, asparagine,phenylalanine, leucine, tryptophan and lysine. In certain embodiments,the destabilising amino acid is arginine. In some proteins, theamino-terminal end is obscured as a result of the protein's conformation(i.e., its tertiary or quaternary structure). In these cases, moreextensive alteration of the amino-terminus may be necessary to make theprotein subject to the N-end rule pathway. For example, where simpleaddition or replacement of the single amino-terminal residue isinsufficient because of an inaccessible amino-terminus, several aminoacids (including lysine, the site of ubiquitin joining to substrateproteins) may be added to the original amino-terminus to increase theaccessibility and/or segmental mobility of the engineered aminoterminus.

Modification or design of the amino-terminus of a protein can beaccomplished at the genetic level. Conventional techniques ofsite-directed mutagenesis for addition or substitution of appropriatecodons to the 5′ end of an isolated or synthesised antigen-encodingpolynucleotide can be employed to provide a desired amino-terminalstructure for the encoded protein. For example, so that the proteinexpressed has the desired amino acid at its amino-terminus theappropriate codon for a destabilising amino acid can be inserted orbuilt into the amino-terminus of the protein-encoding sequence. Wherenecessary, a nucleic acid sequence encoding the amino-terminal region ofa protein can be modified to introduce a lysine residue in anappropriate context. This can be achieved most conveniently by employingDNA constructs encoding “universal destabilising segments”. A universaldestabilising segment comprises a nucleic acid construct which encodes apolypeptide structure, preferably segmentally mobile, containing one ormore lysine residues, the codons for lysine residues being positionedwithin the construct such that when the construct is inserted into thecoding sequence of the protein-encoding polynucleotide, the lysineresidues are sufficiently spatially proximate to the amino-terminus ofthe encoded protein to serve as the second determinant of the completeamino-terminal degradation signal. The insertion of such constructs intothe 5′ portion of a protein-encoding polynucleotide would provide theencoded protein with a lysine residue (or residues) in an appropriatecontext for destabilisation.

In other embodiments, the polypeptide of interest is modified to containa PEST region, which is rich in an amino acid selected from proline,glutamic acid, serine and threonine, which region is optionally flankedby amino acids comprising electropositive side chains. In this regard,it is known that amino acid sequences of proteins with intracellularhalf-lives less than about 2 hours contain one or more regions rich inproline (P), glutamic acid (E), serine (S), and threonine (1) as forexample shown by Rogers et al. (1986, Science 234 (4774): 364-368).

In still other embodiments, the polypeptide of interest is conjugated toa ubiquitin or a biologically active fragment thereof, to produce amodified polypeptide whose rate of intracellular proteolytic degradationis increased, enhanced or otherwise elevated relative to the unmodifiedpolypeptide.

The constructs of the present invention, which contain RNA and/orprotein-destabilising sequences, are particular useful for assaying theactivity of gene expression-modulating elements (GEMEs) including, butnot limited to, transcriptional control elements and cis-actingregulatory elements. These assays provide a more ‘real-time’ analysis ofGEME activity than provided by existing assays and typically comprise:(1) expressing at least in part from a GEME of interest a reporterpolynucleotide operably linked to a RNA destabilising element in a testconstruct and (2) measuring the level or functional activity of thereporter polypeptide produced from the test construct. Generally,control assays are also performed using a control construct comprising aGEME that is different than the GEME of interest. In these embodiments,it is desirable that the reporter polypeptide produced from the testconstruct is detectably distinguishable from the reporter polypeptideproduced from the control construct. The test construct and the controlconstruct may be in the form of separate constructs or a single chimericconstruct. They may also be contained within a single cell or withindifferent cells.

The constructs of the present invention are also useful in screening fordrugs or treatments that alter the activity of geneexpression-modulating elements including transcriptional controlelements (e.g., promoters) and cis-acting regulatory elements (e.g.,enhancers). Compared to existing constructs, a near “real-time”measurement of drug action can be obtained. Accordingly, yet anotheraspect of the present invention contemplates methods for identifying anagent that modulates the activity of a GEME of interest. These methodsgenerally comprise expressing at least partly under the control of theGEME of interest in a test construct a reporter polynucleotide operablylinked to a RNA stability modulating element in the presence and absenceof a test agent The level or functional activity of the reporterpolypeptide in the presence and absence of the test agent is thenmeasured and compared. A difference between the level or functionalactivity of the reporter polypeptide in the presence and absence of thetest agent indicates that the test agent modulates the activity of theGEME of interest. Generally, control constructs are also used in theseassays, which comprise a GEME that is different than the GEME ofinterest. In these embodiments, the reporter polypeptide produced fromthe test construct is suitably detectably distinguishable from thereporter polypeptide produced from the control construct. The testconstruct and the control construct may be in the form of separateconstructs or a single chimeric construct. They may also be containedwithin a single cell or within different cells. In some of theseembodiments, the test construct may be contained within a first celltype or exposed to a first condition and within a second cell type orexposed to a second condition, wherein a difference in the level orfunctional activity of the polypeptide in the presence of the test agentbetween the cell types or conditions provides information on the effectof the test agent on those cell types or conditions (e.g., mode ofaction or specificity).

In some embodiments, the constructs of the present invention compriseone or more elements in any order selected from the group consisting of:

-   -   (i) a multiple cloning site for introducing a sequence of        nucleotides, which site is suitably cleavable enzymatically or        otherwise biochemically to provide a linearised vector into        which PCR amplification products are clonable directly (e.g., an        Ec1HK1 site);    -   (ii) a reporter gene;    -   (iii) a promoter and/or enhancer for regulating expression of a        transcribable polynucleotide (e.g., the polynucleotide that        encodes the polypeptide);    -   (iv) a polyadenylation sequence;    -   (v) a selectable marker gene; and    -   (vi) an origin of replication.

In certain embodiments, the constructs of the present invention are inthe form of vectors or sets of vectors, particularly but not exclusivelyplasmids, with applications in the study or measurement or monitoring ofgene expression (e.g., promoter or enhancer activity). The vectors aresuitably in the form of prokaryotic or eukaryotic vectors. Many othervectors could also be used such as for example viruses, artificialchromosomes and other non-plasmid vectors.

In some embodiments, pairs or sets of plasmids are provided, eachcontaining one or more of the RNA destabilising sequences describedabove in operable linkage with a polynucleotide encoding a destabilisedreporter protein such as, for example, d1EGFP, d1EYFP, d1ECFP ord1HcRed. One plasmid (the control) from each pair or set contains apromoter 5′ of the reporter encoding region. The promoter comprises oneor more elements which are modulatable (i.e., inducible or repressible)by exogenous treatments (e.g., the TRE combined with a minimal promotersuch as mCMV; see FIG. 2 c). Alternatively, a constitutively activepromoter such as TS, SV40, CMV, TK or RSV is used (see FIG. 2 b). Inplant systems the Top-ten promoter could replace TRE, and the 35Spromoter of cauliflower mosaic virus can replace SV40 etc. Agrobacteriumtumefaciens can be used in plants to facilitate gene transfer. The otherplasmid(s) in the pair or set are identical to the control plasmid,except that a cloning site (MCS) replaces the promoter, and the reporterencoding region encodes a reporter similar to but distinguishable fromthe control reporter (see FIG. 2 a). In some embodiments, the controlplasmid encodes a destabilised variant of EGFP (e.g., d1EGFP, d1EYFP ord1ECFP) and the other vectors (test vectors) each encode a differentcolour variant from the same list or d1HcRed or other destabilisedfluorescent protein (same protein half-life). In other embodiments, acontrol and one of the test reporters are incorporated into a singlevector, such as for example a bi-directional plasmid (see FIG. 3).

In the above embodiments, both control and test plasmids encode adestabilised mRNA, which in turn encodes a destabilised protein. Thusthe time lag between decreased promoter activity and decreased reporterprotein levels is significantly reduced compared to the time lag withexisting constructs. Similarly, increased promoter activity is morereadily and quickly detectable due to the reduced levels of pre-existingmRNA and protein. Other differences between the control and testconstructs, which can lead to errors, are minimised by using fluorescentproteins that differ from each other by only a few small mutations.Compared to luciferase or other enzyme based assays, the fluorescentreporters described here, offer several other advantages including:

-   -   Several different reporters can be measured in the same        cells/samples.    -   Live cells can be measured, allowing multiple time points of the        same samples or further manipulation post-measurement e.g.,        measurement of the same cells before and after treatment with a        drug.    -   Successfully transfected cells can be visualised by fluorescent        microscopy. Therefore poor transfections can be identified        simply by looking at the cells under a microscope, without        further investment of resources.    -   No substrates are required, therefore the method is less        technically demanding, faster, less expensive and more accurate.    -   Both control and test reporter expression can be measured        simultaneously by flow cytometry (see advantages of flow        cytometry below).    -   Embodiments utilising TREs as the control promoter can only be        used in Tet-On or Tet-Off cell lines, but as compared to other        control promoters, exhibit less interference from or to the test        promoter and are less affected by various stimuli used to        examine inducibility of the test promoter. Thus, they provide a        more accurate measurement of transfection efficiency and        relative test promoter activity. Control reporter expression can        be switched on or off as required and used to confirm the lack        of promoter cross-talk or compensate for it if present.    -   In other embodiments, the control and one of the test reporters        described above are both incorporated into a single vector,        preferably a bi-directional plasmid. Interference between the        two promoters, which is a major drawback of previous dual        promoter vectors, is minimised by using TREs in the control        promoter and/or insulator DNA (U.S. Pat. No. 5,610,053). Such a        single vector system reduces the inaccuracies of co-transfection        studies.

The invention also provides constructs in which informative promoters orenhancers or their fragments are placed upstream of a reporterpolynucleotide. Informative promoters include, but are not restrictedto, cell cycle-dependent promoters (e.g., cyclin A, B, or D1, histone ortopoisomerase I promoters), promoters activated by apoptotic (celldeath) pathways and promoters/fragments linked to mitogenic signals(Table 1). Examples of informative enhancers that can be used includeany of those used in Clontech's Mercury Pathway Profiling Systems.Clontech's Mercury In Vivo Kinase Assay Kits represent another exampleof how the present invention can be used. In this example the promoterelement is a TRE that is combined in cells with a chimericTetR-transactivator protein that permits transcription from the TRE onlywhen a specific kinase is active and can phosphorylate thetransactivator domain of the fusion protein. Thus, the present inventioncan be used to provide a more real-time measurement of specific kinaseactivity.

Even still another aspect of the present invention contemplates a celltransfected or transduced with a construct or vector as describedherein.

In some embodiments, the constructs or construct-containing cells areintroduced into an organism to allow measurement of reporter activity invivo. Methods for introducing foreign nucleic acid into the nucleome ofan organism or for introducing cells into an organism are known to thoseof skill in the art. In some embodiments of this type, destabilisedluciferase rather than destabilised EGFP variants may be the preferredreporter. For example, transgenic mice expressing destabilisedluciferase under the control of an informative promoter, can be used tomeasure the activity of that promoter in the tissues of a live mouse,using a photon camera (photon camera analysis is described by Contag, etal., 1997). The RNA destabilising sequences serve to improve thetemporal correlation between promoter activity and reporter levels, thusproviding a significant improvement to applications such as drugscreening, which benefit from a near real-time measurement of promoteractivity.

In some applications it is desirable to express, either in vitro incell-based systems or in vivo in mammalian systems, both a reportermolecule and a functional gene product. This may involve two separatemRNAs, each containing a mRNA destabilising element. Alternatively, mRNAdestabilising elements may be incorporated into a single destabilisedtranscript that gives rise to two separate proteins (e.g., using aninternal ribosome entry site; IRES) or a fusion protein comprised of thereporter and the functional gene product.

The invention also provides cell lines stably expressing the constructsof the invention (with or without a control). Such cells haveapplications in areas such as drug screening. For example, cellscontaining a MAPK-dependent reporter vector provide a rapid andinexpensive method for testing the efficacy of drugs designed to inhibitMAPK or any pathway upstream of MAPK-dependent transcription in thosecells. In SKBR3 human breast cancer cells, for example, MAPK activity isdependent on signalling from the overexpressed ErbB2 protein. Therefore,drugs that inhibit ErbB2, would cause a decrease in the fluorescence ofSKBR3 cells containing such a construct but not in cells lacking ErbB2.Alternatively, cells could be tested±drug and±a specific ligand ortreatment that leads to MAPK activation via a different pathway, inorder to monitor inhibition of that pathway. Cell lines (or organisms)stably expressing a vector linked to a cell-cycle-regulated promoter canbe used as very fast, simple and inexpensive means for measuringcell-cycle progression or cell proliferation. Such cell lines haveobvious utility in drug screening and are contemplated in the presentinvention. Examples of cell-cycle regulated promoters are readilyavailable, for example, (Lee, H et al. 1995), (Stein, J et al. 1996) and(Huet, X et al. 1996).

Other embodiments of the present invention are directed to constructsfor the study of post-transcriptional regulation, particularly mRNAstability. These constructs typically comprise a reporter polynucleotideoperably linked to a transcriptional control element. In illustrativeexamples of these constructs, the reporter polynucleotide encodes adestabilised reporter protein, such as but not limited to a destabilisedvariant of EGFP (e.g., d1EGFP, d1EYFP, d1ECFP), with a different colourvariant in each separate vector. The TRE (linked to a minimal promotersuch as mCMV) is 5′ of the reporter encoding region and drivestranscription in a tetracycline (or doxycycline) dependent fashion.Other inducible promoter systems can also be used.

Typically the constructs for studying post-transcriptional regulationcomprise sites for inserting known or suspected post-transcriptionalcontrol elements. In some embodiments, the RNA destabilising elementsdescribed above are not included and in their place, MCSs are located,primarily in the 3′-UTR (see FIG. 4 a) but also in the 5′-UTR and/orcoding region. In some embodiments, sequences thought to affect mRNAstability can be tested by cloning them into the appropriate cloningsite of a construct containing one colour variant and measuring the rateof decrease in reporter levels after blocking transcription withtetracycline or doxycycline (see FIG. 7). If desired, the rate of decaycan be compared between the “test construct” and the “controlconstruct,” (which suitably encodes a different colour reporter proteinand does not contain the sequence being tested) in the same cells. TheMCS may usefully comprise or work in conjunction with restrictionendonuclease sites which allow direct cloning of PCR products havingoverhangs (see below).

In related embodiments, the invention provides methods for assaying theactivity of a post-transcriptional control element or for identifying apost-transcriptional control element or for identifying an agent thatmodulates elements of this type. These methods generally comprise: (1)expressing from a transcriptional control element in a test construct areporter polynucleotide that is operably linked to a nucleic acidsequence that encodes, or is suspected to encode, a post-transcriptionalcontrol element; and (2) measuring the level or functional activity ofthe reporter polypeptide produced from the test construct. Often thesemethods will include the use of control constructs, which do notcomprise the nucleic acid sequence that encodes, or is suspected toencode, the post-transcriptional control element. The control constructsmay comprise the same or different transcriptional control element asthe test construct. In these embodiments, the reporter polypeptideproduced from the test construct is suitably detectably distinguishablefrom the reporter polypeptide produced from the control construct. Thetest construct and the control construct may be in the form of separateconstructs or a single chimeric construct. They may also be containedwithin a single cell or within different cells. In some embodiments, thetranscriptional control element is modulatable, including inducible orrepressible promoters. In these embodiments, the methods desirablyfurther comprise (1) inducing or repressing the transcriptional controlelement of the test construct, and optionally of the control construct;and (2) measuring changes in the level or functional activity of thereporter polypeptide produced from the test construct, and optionallyfrom the test construct, over time.

In certain embodiments, which are directed to identifying agents thatmodulate a post-transcriptional control element of interest, theexpression of the reporter polynucleotide is carried out in the presenceand absence of a test agent and the levels or functional activities ofthe reporter polypeptide produced in the presence and absence of thetest agent are compared. A difference between the level or functionalactivity of the reporter polypeptide in the presence and absence of thetest agent indicates that the test agent modulates the activity of thepost-transcriptional control element. In some of these embodiments, thetest construct may be contained within a first cell type or exposed to afirst condition and within a second cell type or exposed to a secondcondition, wherein a difference in the level or functional activity ofthe polypeptide in the presence of the test agent between the cell typesor conditions provides information on the effect of the test agent onthose cell types or conditions (e.g., mode of action or specificity).

In other related embodiments, one or more RNA destabilising element(s)are included to assist scientists specifically searching for RNAstabilising elements. RNA stabilising elements are useful for increasinglevels of expressed protein for example during protein purificationwhere high levels or protein are required or when, a promoter is weak.Similarly, other embodiments include RNA stabilising element(s) toassist scientists specifically searching for RNA destabilising elements.

In still other embodiments, the control and one of the test reportersare both incorporated into a single vector, desirably a bi-directionalplasmid (see FIG. 4 b). Interference between the two promoters andmoreover, transcription effects of the element or various stimulitested, is circumvented by using a TRE or similar element to drive bothreporters and by measuring reporter levels after addition of doxycycline(or tetracycline), which shuts off transcription from the vector.

A related aspect of the present invention extends to a geneticallymodified non-human organism comprising a construct as broadly describedabove. Accordingly, the present invention is directed towardsgenetically modified animals that contain one or more constructs of theinvention in their nucleomes, and especially in their genomes. Thegenetic modification is generally in the form of a transgene and thusthe genetically modified animal of the present invention is a transgenicanimal that comprises at least one transgene in its cells, whichincludes a construct as broadly described above. The transgene issuitably contained within somatic cells of the animal, although it mayalso be contained within its germ cells. Usually, the transgenic animalis a mammal, which is suitably selected from the order Rodentia. In someembodiments, the transgenic mammal is a mouse, although rats are also ofparticular utility. However, it will be understood that the presentinvention is not restricted to these species. For example, thetransgenic animal may be a goat, cow, sheep, dog, guinea pig or chicken.

The genetically modified animals of the present invention may beprepared by any number of means. In one method, a nucleic acid targetingconstruct or vector is prepared comprising two regions flanking thetransgene wherein the regions are sufficiently homologous with portionsof the genome of an animal to undergo homologous recombination withthose portions. Alternatively, constructs for random integration neednot include regions of homology to mediate recombination. Conveniently,markers for positive and negative selection are included in theconstructs to permit selection of recombinant host cells. The targetingDNA construct is generally introduced into an embryonic stem (ES) cellor ES cell line. Methods for generating cells having gene modificationsthrough homologous recombination are known in the art.

In order that the invention may be readily understood and put intopractical effect, particular preferred embodiments will now be describedby way of the following non-limiting example.

EXAMPLES Example 1 Cloning DNA Elements into Vectors

Cloning is carried out according to existing methods, using restrictionenzyme sites in the MCS or direct ligation of PCR products in the caseof vectors with a “T overhang” in the MCS. With respect topost-transcriptional reporter vectors, however, the inclusion of a MCSin the 3′-UTR or other regions is a significant improvement over currentvectors, which were designed for transcriptional or other studies and donot contain convenient cloning sites in these locations.

Example 2 Transfection

Co-transfection of control and test vectors is performed as per existingmethods (e.g., Fugene [Boehringer Mannheim, Mannheim, Germany] orelectroporation), except in the case of the single (e.g.,bi-directional) vector systems described above, which require only onevector and thus eliminate inaccuracies associated with co-transfection

Example 3 Measurement of Reporter Expression

An immediate advantage of the vGFP system is that reporter expressioncan be visualized directly in living cells, simply by viewing the tissueculture plate or flask under a fluorescent microscope. Therefore, poortransfections can be identified and discarded before any additional timeis wasted. Quantitative measurement can be performed using a fluorometer(e.g., 96 well plate format) and since live cells can be measured, thesame samples can be measured repeatedly e.g., in a time course.

A further advantage compared to luciferase and other enzyme based assaysis that flow cytometry can also be used to measure reporter levels.

Example 4 Advantages of Using Flow Cytometry to Measure Reporter Levels

-   -   (i) Two or more reporters (control and test) as well as        additional parameters, can be measured individually in every        cell at a rate of >2,000 cells per second. Therefore, in this        application, the method yields thousands to hundreds of        thousands of data points per sample versus one datum point for        existing luciferase assays.    -   (ii) Accurate measurement of transfection efficiency: This is        useful for optimising transfection protocols. In addition to        allowing comparison of different methods, it is also possible to        measure both expression per cell and the proportion of cells        expressing. This helps the investigator to determine the cause        of any problems.    -   (iii) Identification of co-transfection errors: Co-transfection        studies are based on the premise that cells will take up and        express an amount of control reporters, which is proportional to        the amount of test plasmid taken up by the same cells. This is        not always the case. By using the flow cytometry method        described here, it is possible to correlate test versus control        expression levels in different cells of the same sample. Invalid        samples can be identified by the lack of a good linear        relationship between test and control reporter levels. Such        errors go unnoticed in current methods.    -   (iv) Simultaneous measurement of additional parameters:        Fluorescent labelled antibodies can be used to quantify specific        proteins on a cell by cell basis and this can be correlated with        reporter levels to determine whether that protein affects gene        expression via the element cloned into the reporter construct.        Alternatively, the protein of interest can be expressed as a        vGFP-fusion protein (the protein of interest fused to a GFP        variant) via transfection of an appropriate expression vector        (inducible or non-inducible). Levels of the specific protein can        then be correlated with the expression of a different GFP        variant linked to a regulatory element of interest        (co-transfected or transfected at a different time). In a third        application, the vGFP reporter is linked to regulatory elements        (e.g., promoters) thought to be cell cycle specific. Transfected        cells are stained with a fluorescent DNA dye such as propidium        iodide to measure DNA content, which is then correlated with        reporter expression. In principle, several of the DNA constructs        described herein, each containing a different vGFP, could be        co-expressed and independently measured. Furthermore, other        fluorescent markers could be used in conjunction with these        vectors (singly or in multiples).    -   (v) Cell Sorting: Using a cell sorter, it is possible to isolate        viable vGFP expressing cells from the non-expressors. This        technique can be used to select stably expressing cells or to        remove non-expressors prior to assay initiation. Similarly, it        is possible to remove cells expressing very low and/or very high        levels of vGFP. This can be used to generate a more homogeneous        population and/or to remove cells expressing levels so high that        they may not be physiological relevant or may perturb normal        cellular function and/or may otherwise adversely affect the data        obtained from the DNA vectors described herein.

It is important to note that transient and stable transfections ofexpression vectors result in a cell population with very heterogeneouslevels of expression. In general a thousand fold difference between thehighest and lowest expresser is not unusual. The present invention notonly offers a method for selecting homogeneous populations when required(see v above), but can also utilise heterogeneity to the benefit of thescientist. For example, identifying co-transfection errors. Anotherexample of this relates to (iv) above. To determine whether protein Xaffects transcription from promoter Y, then cells are transfected with areporter construct expressing d1EGFP under the control of promoter Y. Ifrequired, cell sorting can be used to isolate cells transiently orstably expressing appropriate levels of d1EGFP. These cells are in turntransiently transfected with a vector expressing a protein X-EYFP fusionprotein. During flow cytometry, EGFP is plotted on one axis and EYFP onthe other. A positive correlation would indicate that protein Xincreases transcription from promoter Y and a negative correlation wouldindicate that protein X inhibits transcription from promoter Y.

Currently, scientists attempting to establish such a correlation wouldselect several different clones of high versus low expressors of proteinX. Each clone would then be separately transfected with a promoterY-luciferase construct and the luciferase activities measured. The useof cell clones requires months of preparation and introduces manyvariables including pre-existing heterogeneity amongst the host cellsand variable sites of vector integration (vector DNA may interfere witha specific gene at the integration site and this site is different forevery clone). Furthermore, such a method yields very few data points,with each datum point obtained from a different transfection of adifferent clone. Thus, the new system is not only more versatile but isquicker and more accurate than existing methods.

Example 5 Laser Scanning Cytometry (LSC)

Unlike flow cytometry, LSC measures multi-colour fluorescence and lightscatter of cells on slides, and records the position and time ofmeasurement for each cell analysed. This technique provides dataequivalent to flow cytometry but has the advantage of being microscopeslide based (Darzynkiewicz et al., 1999; Kamentsky et al., 1997). Owingto the fluorescence of GFP and its variants, the techniques describedfor flow cytometry are also applicable to LSC.

Example 6 Specific Methods for Post-Transcriptional Assays

These are best summarised by using the example of a study aimed atdetermining whether a specific 3′-UTR fragment affects mRNA stability.Although this example is one of transient expression, stabletransfection could also be used.

-   -   (i) The 3′-UTR fragment is ligated into the 3′-UTR cloning site        of the test vector and co-transfected with the control vector        into a Tet-Off cell line. In the case of the bi-directional        vector, no control vector is required. Indeed, the typical        application does not require a control reporter or vector since        rate of decay can be measured in samples from within the same        transfection. 5′-UTR fragments can be tested by inserting them        into vectors with a 5′-UTR cloning site.    -   (ii) The cells are grown in the absence of doxycycline (or        tetracycline) for 6-48 h to allow expression of both vectors.        Alternatively, cells are grown with low doses of doxycycline (or        tetracycline), for 6-48 h to block transcription and then        switched to medium without doxycycline (or tetracycline) for        2-12 h to provide a brief burst of transcription.    -   (iii) High doses of doxycycline (or tetracycline) are then        applied to shut off transcription from both vectors.    -   (iv) The fluorescence of both reporters is measured (by flow        cytometry, fluorometry or LSC) in a time course following        addition of doxycycline (or tetracycline).

If the cloned element confers mRNA instability, a more rapid decrease in“test” fluorescence will be seen compared to “control” fluorescence ofthe same cells or sample. Similar studies can be used to test a mRNAelement's response to certain stimuli or its effect in different cellsor cells expressing different amounts of a specific protein, such as aRNA-binding protein. Applying the stimulus after doxycycline willdetermine whether pre-existing transcripts are affected by the stimulus.Inserting the element in different locations (e.g., 5′-UTR, 3′-UTR) willdetermine whether its function is dependent on position. Inserting aprotein/polypeptide coding sequence (in frame) within thereporter-coding region of the vector, can be used to determine theeffect of that sequence on mRNA and protein stability.

RNA can be extracted from transfected cells and used to measure reportermRNA directly.

Example 7 Transcription Reporter Vectors

The vectors are plasmids suitable for expansion in E. coli andexpression of a fluorescent reporter in eukaryotic cells. The plasmidsmay be used in sets. Each set is comprised of one or more “control”vectors and one or more “test” vectors. Every vector within a setexpresses a similarly destabilised mRNA and a similarly destabilisedfluorescent reporter protein. In addition to the standard features ofsuch plasmids (ampicillin resistance, origin of replication etc.), eachplasmid contains the following construct (see also FIGS. 2 and 3):

-   -   5′-MCS/promoter-transcription start site-5′-UTR-ATG-vEGFP        encoding region-stop codon-3′-UTR with mRNA destabilising        element-polyadenylation signal    -   Where:    -   MCS/promoter denotes either a multiple cloning site (test        vectors; see FIG. 2 a) or a constitutively active promoter such        as SV40 (control vectors; see FIG. 2 b) or an inducible promoter        such as TRE-mCMV (control vector; see FIG. 2 c).    -   ATG denotes a translation start codon.    -   Stop codon denotes a translation stop codon.    -   5′-UTR denotes a 5′ untranslated region.    -   3′-UTR with mRNA destabilising element denotes a 3′ untranslated        region containing one or more of the mRNA destabilising elements        outlined.    -   vEGFP denotes a destabilised variant of EGFP. One set of        plasmids is provided for each type of destabilising modification        (e.g., 1 hr half-life, 2 hr half-life). Within each set of        plasmids, one vector is provided for each different colour        variant. For example, one set contains vectors expressing        d1EGFP, d1EYFP, d1ECFP whereas another set expresses the d2        variants.

In other examples, the control and one of the test reporters describedabove are both incorporated into a single vector, preferably abi-directional plasmid (see FIG. 3).

Example 8 Post-transcription Reporter Vectors

Similar to the transcription reporter “control” vectors that contain aTRE-mCMV promoter, except that the mRNA destabilising element in the3′-UTR is replaced with a MCS (see FIG. 4 a). In some embodiments, MCSare also located in the 5′ UTR and/or coding region.

Such a construct can be used as a “test” or a “control” vector for thepost-transcriptional assays outlined herein.

In other examples, the control and one of the test reporters describedabove are both incorporated into a single vector, preferably abi-directional plasmid (see FIG. 4 b).

Example 9 Reporter Vectors for Assaying Specific Pathways

Vectors similar to those described herein, into which a regulatoryelement has been inserted into the MCS for the purpose of studying ormeasuring the function of said regulatory element. For example, plasmidssimilar to the transcription reporter plasmids outlined herein, exceptthat they contain within the MCS, a promoter or promoter element(s) orenhancer(s) that are responsive to pathways such as those referred to inTable 1 and/or contain any of the following cis-acting enhancer elementsas described in Clontech's Mercury Pathway Profiling Systems: AP1, CRE,E2F, GRE, HSE, ISRE, Myc, NFAT, NFκB, p53, Rb, SRE. The reporter ispreferably a destabilised version of GFP, luciferase or SEAP.

Cell lines and Mice for Assaying Specific Pathways

Cell lines or genetically modified mice stably expressing one or more ofthe vectors described herein.

Example 10 Method of Use

The vectors described in this invention are used for experimentation inessentially the same manner as the existing vectors that they replace,with the exception of the new methods described herein.

Method of Construction

The vectors and DNA constructs outlined here are assembled usingstandard cloning techniques. The SV40 and TRE-mCMV promoters describedhere as well as the more standard components of plasmid vectors (e.g.,origin of replication, antibiotic resistance or another selection gene)are readily available in a variety of common vectors. DNA sequencesencoding the destabilised variants of EGFP (e.g., d1EGFP, d1EYFP, d1ECFPand d2EGFP, d2EYFP, d2ECFP) are available from Clontech (ClontechLaboratories Inc., Palo Alto, Calif., USA). DNA sequences encodingdestabilised DsRed variants are constructed by fusing to the 3′ end ofthe DsRed encoding region, sequences encoding the degradation domains(or mutants thereof) from short-lived proteins. For example, amino acids422-461 from mouse ornithine decarboxylase, which contains a PESTsequence. Such sequences could potentially be derived from existingdEGFP variants.

Example 11 Summary

In summary the present vectors and methods are now available:

-   -   Expression vectors or parts thereof that incorporate one or more        mRNA instability elements in order to provide a relatively        short-lived mRNA. Compared to existing expression vectors, the        vectors claimed here provide kinetics of protein expression that        correlate more closely with promoter activity. For example, the        time lag between decreased promoter activity and decreased mRNA        and protein levels is substantially reduced.    -   Expression vectors or parts thereof encoding a destabilised mRNA        that in turn, encodes a destabilised protein. Compared to        existing vectors, the vectors claimed here provide kinetics of        protein expression that correlate more closely with promoter        activity.    -   Expression vectors or parts thereof in which the mRNA        destabilising elements are comprised of sequences cloned from        short-lived mRNAs such as c-fos, examples of short-lived mRNAs        include; c-fos, c-myc, GM-CSF, IL-3, TNF-alpha, IL-2, IL-6,        IL-8, Urokinase, bcl-2, SGLT1 (Na(+)-coupled glucose        transporter), Cox-2 (cyclooxygenase 2), IL8, PAI-2 (plasminogen        activator inhibitor type 2), beta1-adrenergic receptor, GAP43        (5′-UTR and 3′-UTR) AU-rich elements (AREs) and/or U-rich        elements, including but not limited to single, tandem or        multiple or overlapping copies of the nonamer UUAUWUA(U/A)(U/A)        [SEQ ID NO:2] (where U/A is either an A or a U) (Lagnado et        al 1994) and/or the pentamer AUUUA [SEQ ID NO:3] (Xu et al 997)        and/or the tetramer AUUU [SEQ ID NO:4] (Zubiaga et al. 1995).        Also included are minor modifications to or permutations of the        elements listed above. The term “tandem copies,” allows for both        duplication and/or non-duplication of one or more of the outer        nucleotides. For example, tandem copies of the pentamer AUUUA        [SEQ ID NO:3], includes sequences such as AUUUAUUUAUUUA [SEQ ID        NO:5] as well as AUUUAAUUUAAUUUA [SEQ ID NO:6]. The 3′-UTR or        5′-UTR regions of short-lived mRNAs often contain destabilising        sequences.    -   Expression vectors or parts thereof in which the mRNA        destabilising elements were identified or validated using the        vectors described herein, which provide substantially improved        methods for identifying such elements.    -   Expression vectors or parts thereof, in which the destabilised        mRNA encodes a short-lived reporter protein such as a        destabilised variant of EGFP or luciferase. Compared to existing        reporter vectors, the vectors claimed here provide kinetics of        reporter expression that correlate more closely with promoter        activity. For example, the time lag between decreased promoter        activity and decreased mRNA and protein levels is substantially        reduced.    -   Sets of reporter vectors or parts thereof that encode similarly        destabilised mRNAs (similar to other vectors in the same set),        which in turn, encode similarly (similar to other vectors in the        same set) destabilised variants of EGFP or DsRed or other        fluorescent markers. One or more vectors (control vectors)        within each set contain a constitutive promoter (e.g., SV40,        CMV, RSV, TK, TS; see FIG. 2 b) or an inducible promoter (e.g.,        TRE-mCMV; see FIG. 2 c), whereas the other vectors (test        vectors) within each set contain a cloning site (e.g., MCS) in        place of the promoter (e.g., see FIG. 2 a). Applications of        these vectors include but are not limited to the study or        measurement of promoter activity. For example, a promoter        element of interest can be cloned into the MCS of a test vector        encoding d1EGFP and reporter expression measured relative to        that of a control vector expressing d1EYFP. Also claimed is each        individual vector described well as bi-directional vectors or        other single vector systems that incorporate one test and one        control reporter construct within the same vector (e.g., FIG. 3        a and FIG. 3 b). Compared to existing sets of reporter vectors,        the vector sets claimed here offer the following advantages:        -   (a) A measurement of promoter activity that is closer to            real-time.        -   (b) Decreased errors due to the closer similarity between            control and test constructs.        -   (c) Decreased errors resulting from cross talk between test            promoters and the control promoters. By utilising inducible            promoters in the control vectors, such cross talk is            minimised and/or identified and corrected for via            measurement with and without induction.        -   (d) Can be used in conjunction with the flow cytometry/LSC            methods described.    -   Reporter vectors or sets of reporter vectors or parts thereof        that utilise an inducible promoter, preferably but not        exclusively the tetracycline responsive element (TRE), to drive        expression of a destabilised fluorescent reporter protein        (preferably but not exclusively destabilised EGFP variants).        Such vectors contain cloning sites in the 3′-UTR (e.g., FIG. 4        a) and/or 5′-UTR and/or reporter coding region, such that        regulatory elements or putative regulatory elements can be        cloned into a vector expressing one color fluorescent reporter        and, if required, compared to a control vector which expresses a        different color reporter and does not contain the element of        interest. Such vectors have applications in the study or        measurement of post-transcriptional regulation, since        transcription can be shut off as desired via the inducible        promoter. The advantages offered by these vectors include those        listed in b-d, the ability to separate post-transcriptional        effects from transcriptional effects and also:        -   (a) incorporation of convenient cloning sites, not present            in other vectors; and        -   (b) the technique is more rapid than any existing method.    -   Single vector systems that essentially link one test and one        control construct and described (e.g., FIG. 4 b). Both test and        control reporters are driven by an inducible promoter and the        cloning sites allow ligation of regulatory elements into the        test construct only. In addition to the advantages of vectors        outlined, the single vector systems eliminate problems and        inaccuracies associated with co-transfection of separate test        and control vectors.    -   The use of flow cytometry or LSC to measure the levels of 2 or        more fluorescent reporters expressed via the vectors outlined.        In this application, the method yields thousands to hundreds of        thousands of data points per sample versus one datum point for        existing enzyme-based assays. Two or more reporters (control and        test) as well as additional parameters (e.g., DNA content,        levels of other proteins) can be measured individually in every        cell. Also encompassed is the use of flow cytometry to correlate        the levels of 2 or more reporters in multiple cells within the        same sample and the utilisation of such data to optimise        transfection protocols and/or identify problems associated with        co-transfection. For example, invalid samples can be identified        by the lack of a good linear relationship between test and        control reporter levels. Such errors go unnoticed in current        methods.    -   Methods for utilising the post-transcriptional reporter vectors        claimed. These methods are best summarised by using the example        of a study aimed at determining whether a specific 3′-UTR        fragment affects mRNA stability. Although this example is one of        transient expression, stable transfection could also be used.        -   (i) The 3′-UTR fragment is ligated into the 3′-UTR cloning            site of the test vector and co-transfected with the control            vector into a Tet-Off cell line. In the case of the single            vector system, no control vector is required. 5′-UTR            fragments can be tested by inserting them into vectors with            a 5′-UTR cloning site.        -   (ii) The cells are grown in the absence of doxycycline (or            tetracycline) for 6-48 h to allow expression of both            vectors. Alternatively, cells are grown with low doses of            doxycycline (or tetracycline), for 6-48 h to block            transcription and then switched to medium without            doxycycline (or tetracycline) for 2-12 h to provide a brief            burst of transcription.        -   (iii) High doses of doxycycline (or tetracycline) are then            applied to shut off transcription from both vectors.        -   (iv) The fluorescence of both reporters is measured (by flow            cytometry, fluorometry or LSC) in a time course following            addition of doxycycline (or tetracycline).    -   If the cloned element confers mRNA instability, a more rapid        decrease in “test” fluorescence will be seen compared to        “control” fluorescence of the same cells or sample. Similar        studies can be used to test a mRNA element's response to certain        stimuli or its effect in different cells or cells expressing        different amounts of a specific protein, such as a RNA-binding        protein. Applying the stimulus after doxycycline will determine        whether pre-existing transcripts are affected by the stimulus.        Inserting the element in different locations (e.g., 5′-UTR,        3′-UTR) will determine whether its function is dependent on        position. Inserting a protein/polypeptide coding sequence (in        frame) within the reporter protein-coding region of the vector        can be used to determine the effect of that sequence on mRNA and        protein stability.    -   RNA can be extracted from transfected cells and used to measure        reporter mRNA directly.    -   Cell lines transiently or stably expressing one or more of the        expression constructs or parts thereof claimed.    -   Cell lines transiently or stably expressing one or more of the        expression constructs or parts thereof claimed, wherein the        expression construct contains a regulatory element that serves        as a marker for the activation of signal transduction pathways        associated with human disease and/or response to drug treatment.        Such pathways include, but are not restricted to the list in        Table 1 and those indicated elsewhere in this document (e.g.,        CRE, SRE, AP1, cyclin A, B and D1 promoters).    -   Transgenic mice, knock-in mice or other genetically modified        mice expressing one or more of the expression constructs or        parts thereof claimed.    -   Transgenic mice, knock-in mice or other genetically modified        mice expressing one or more of the expression constructs or        parts thereof claimed, wherein the expression construct contains        a regulatory element that serves as a marker for the activation        of signal transduction pathways associated with human disease        and/or response to drug treatment. Such pathways include, but        are not restricted to the list in Table 1.    -   Destabilised variants of DsRed or the mutant DsRed1-E5. These        can be constructed by fusing to the C-terminus of DsRed,        degradation domains (or mutants thereof) from various unstable        proteins. For example, amino acids 422-461 of mouse ornithine        decarboxylase, which contains a PEST sequence (Li et al. 1998).        Additional destabilising elements can also be added. Also        contemplated are DNA constructs encoding destabilised variants        of DsRed.    -   Vectors encoding destabilised variants of DsRed outlined,        including such vectors also containing the mRNA instability        elements outlined.    -   The following method for creating Tet-Off or Tet-On cell lines:    -   The tTA or rtTA expression vector, preferably a retrovirus,        adenovirus or plasmid, is stably expressed in the cell line of        interest using standard techniques and expressing cells are        isolated via a drug resistance marker. These cells are then        transiently transfected with a TRE-vGFP construct and subjected        to several rounds of cell sorting by flow cytometry. For        example, good Tet-Off cells would show no fluorescence in the        presence of doxycycline and are sorted as such. After a further        5-48 hr without doxycycline, green cells are sorted. Finally,        the cells are grown for a week or more without doxycycline and        sorted a final time to eliminate stably transfected (green)        cells.

Example 12 Vectors Incorporating mRNA and Protein-Destabilising Elements

The coding region of interest (e.g., a reporter such as EGFP orluciferase) could include combined sequence of a protein-destabilisingelement (e.g., d1 mutant of MODC; Clontech, but also including otherPEST sequences or other protein-destabilising elements such asubiquitination sites) and a mRNA destabilising element (e.g., AU-richelement).

For example, the stop codon of luciferase and DsRed is replaced with aHind3 site (AAGCTT [SEQ ID NO:7]) to allow the addition of the sequence:AAGCTTAGCCATGGCTTCCCGCCGGCGGTGGCGGCGCAGGATGATGGCACGCTGCCCATGTCITGTGCCCAGGAGAGCGGGATGGACCGTCACCCTGCAGCCTGTGCITCTGCTAG GATCAATGTGTAG[SEQ ID NO:8] which is Clontech's d1 mutant of MODC that confers a 1-hrhalf life to EGFP. This is followed by a linker (which becomes part ofthe 3′-UTR and then: UUAUWUAUU GGCGG UUAUUUAUU CGGCG UUAUUUAUU GCGCGUUAUUUAUU ACUAG, [SEQ ID NO:9] which contains 4 nonamers and connects tothe Xbal site of the parent vector (pGL3; Promega) also in the 3′-UTRbut further downstream.

Example 13 Direct Ligation of PCR Products

Inclusion into the MCS of a vector of two separate but nearby RErecognition sites, which, when cut with that/those RE(s), leave a 3′overhang of a single T nucleotide at both ends of the remaining vector.For example, the recognition sequence for Ec1HK1 is GACNNN, NNGTC [SEQID NO:10] (cuts between 3^(rd) and 4^(th) N from 5′ leaving a 3′overhang of a single N at each end). Two of these sites areincorporating into the MCS, such that the short region between them isreleased by digestion with Ec1HK1, leaving a linearised vector with a 3′overhang of a single N at each end. In this example, the upstreamrecognition sequence should be 5′GACNNTNNGTC3′ [SEQ ID NO:11] and thedownstream sequence 5′GACNNANNGTC3′ [SEQ ID NO:12]. After cutting withEc1HK1, the large vector fragment will contain a single 3′ T overhang atboth ends (similar to Promega's pGEM-T Easy vector). This facilitatesthe direct ligation of PCR products that are produced with a polymerasesuch as Taq, that yields a 5′ A overhang. This constitutes a significantimprovement over standard MCSs, which do not support direct ligation ofPCR products without inclusion of RE sites into PCR primers andsubsequent digestion of PCR product. This is also a significantimprovement over the pGEM-T Easy vector, which cannot be amplified(supplied as linear) and is useful only for subcloning (i.e., PCRproducts are typically ligated into pGEM-T Easy, amplified and thenremoved by RE digestion and subsequently cloned into the expressionvector of interest). Thus, the present MCS permits direct ligation ofPCR products without the need for digesting them with a RE (which isoften problematic) or subcloning them into an intermediate vector.

Example 14 Destabilised Reporter Model Shows Improved Real-Time Analysis

Plasmid reporter vectors were assembled in a pGL3-Basic (Promega)backbone (ampicillin resistance gene etc.) using standard cloningtechniques. A tetracycline-responsive element (IRE), derived fromClontech's pTRE-d2EGFP vector was inserted into the MCS. In someconstructs the luciferase-coding region was replaced with the d1EGFP- ord2EGFP- coding sequence (including Kozak sequence) as defined byClontech. This was achieved by PCR using appropriate primers withconvenient 5′ flanking RE sites. In some constructs, specific examplesof mRNA destabilising elements were cloned into the 3′-UTR-encodingregion. Typically, these sequences were prepared by synthesising andthen hybridising the sense and antisense sequences. Flanking sequencesprovided overhanging “sticky ends” that are compatible with thosegenerated when the 3′-UTR-encoding region is cut with specificrestriction enzymes. Following digestion of the vector with theseenzymes and subsequent purification, the hybridised oligomers wereligated into the vector using standard techniques. PCR of genomic DNA orcDNA from an appropriate source was used as an alternative method forobtaining the larger destabilising elements such as c-myc-ARE. Verysmall elements (e.g., 1 or 2 nonamers) were incorporated into a reversePCR primer that contained a 5′ flanking RE site and a 3′ flanking regioncomplementary to the pre-existing 3′-UTR in the vector template.Following PCR with an appropriate forward primer (complementary to theprotein-coding region and overlapping an endogenous RE site), the PCRproduct was digested with the appropriate RE sites and ligated into theoriginal vector.

Nomenclature:

-   -   B=Vector backbone derived from Promega's pGL3-Basic;    -   T=Tetracycline-responsive element (TRE), derived from Clontech's        pTRE-d2EGFP vector and used as a promoter to drive transcription        of the reporter;    -   G1=GFP with 1 hr half-life used as reporter i.e., d1EGFP protein        encoding sequence as defined by Clontech;    -   G2=GFP with 2 hr half-life used as reporter i.e., d2EGFP protein        encoding sequence as defined by Clontech;    -   L=Luciferase used as reporter i.e., The Firefly luciferase        encoding sequence from pGL3 -Basic (Promega);    -   R=DsRed2 used as the reporter;    -   R1=DsRed fused at the carboxy-end to the same MODC mutant as        present in d1EGFP;    -   N6=6 copies of the nonamer TTATTTATT [SEQ ID NO:13] inserted        into the 3′-UTR-encoding region.    -   N4=4 copies of the nonamer TTATTTATT [SEQ ID NO:13] inserted        into the 3′-UTR-encoding region;    -   N2=2 copies of the nonamer TTATTTATT [SEQ ID NO:13] inserted        into the 3′-UTR-encoding region;    -   N1=1 copy of the nonamer TTATTTATT [SEQ ID NO:13] inserted into        the 3′-UTR-encoding region;    -   fos=The c-fos ARE as defined by Shyu et al (1989) inserted into        the 3′-UTR-encoding region i.e.,        5′AAAACGTTTTATTGTGTTTTTAATTTATTTATTAA        GATGGATTCTCAGATATTTATATTTTTATTTTATTTTTTT3′ [SEQ ID NO:14];    -   myc=the myc ARE defined as follows 5′ATGCATGATCAAATGCAACCTCACA        ACCTTGGCTGAGTCTTGAGACTGAAAGATTTAGCCATAATGTAAACTGCCT        CAAATTGGACTTTGGGCATAAAAGAACTTTTTTATGCTTACCATCTTTTTTT        TTTCTTTAACAGATTTGTATTTAAGAATTGTTTTTAAAAAATTTTAAGATTT        ACACAATGTTTCTCTGTAAATATTGCCATTAAATGTAAATAACTTT3′ [SEQ ID NO:15].

Method:

Five micrograms of maxiprep quality DNA was transfected into ˜50%confluent 10 cm flasks of HeLa Tet-Off cells (Clontech) using Fugenereagent (Roche). Ten hours later, the flasks of cells were each splitinto ˜12 small (6 cm) dishes and then incubated overnight (˜12-14 hrs).At this time point (typically designated time zero or T₀), doxycyclinewas added to the culture media of most plates at a final concentrationof 1 microgram per ml. Cells were trypsinised and collected at this andsubsequent time points. For constructs expressing GFP, these sampleswere analysed by flow cytometry using standard FITC filters. Total GFPfluorescence was measured by gating out non-transfected cells(background fluorescence only) and then multiplying the meanfluorescence per cell (with background fluorescence subtracted) by thenumber of positive cells. RFP fluorescence (DsRed) was measuredsimilarly using appropriate filters. Cells transfected withluciferase-encoding vectors were lysed and measured in a luminometerusing Promega's Dual Luciferase Assay methods and reagents.

Data are typically expressed as the percentage of reporter (fluorescenceor luminescence) remaining, relative to time zero.

Since the doxycycline added at time zero causes a block in transcriptionof the reporter, the rate of decrease in reporter levels indicates thetime lag between altered transcription and altered reporter/proteinlevels. A prime purpose of the invention is to reduce this time lag andFIGS. 7, 8, 9 and 11-14 demonstrate that this is achieved.

As an example of the utility of this invention, a pharmaceutical companymay wish to screen for drugs that reduce transcription of a geneinvolved in disease. The tetracyclineldoxycycline-induced block intranscription from the TRE promoter is a model of such a system. FIGS. 7and 8 show that with the standard luciferase reporter vectors, even atotal block in transcription (with doxycycline) is not detectable as adecrease in luciferase activity within 10 hrs. The destabilised EGFPmutants represent an improvement in that the total block intranscription is detectable as a 50% decrease in EGFP fluorescencewithin 11 hrs (d2EGFP; BTG2) or 7 hrs (d1EGFP; BTG1). However, when thelatter reporter is combined with a mRNA destabilising element such as 4copies of the nonamer UUAUUUAUU [SEQ ID NO:1] (BTG1N4), a 50% decreasein reporter levels is detectable within 3 hrs. It follows that anincrease in a transcription would also be detected sooner withconstructs containing the destabilising elements (Roth, 1995).

Of course the action of doxycycline is not immediate so that part of thetime lag is due to the time required for this drug to induce a 100%transcriptional block. Therefore, the “Effective rate of decay” wasmeasured by plotting data points subsequent to and relative to the timepoint at 4 hrs after addition of doxycycline (FIG. 9). The effectiverate of decay therefore excludes the delay in drug action and is acombined effect of protein and mRNA half-lives. FIG. 9 shows theeffective rate of decay with constructs containing 1, 2 or 4 nonamers.These data show that 4 nonamers are more efficient than 2, which is moreefficient than 1. Furthermore, these data show that by combining a 1 hrhalf-life protein (d1EGFP) with 4 nonamers, an effective rate of decayof approximately 1 hr 20 mins can be achieved. This is very close to theI hr half-life of the protein and demonstrates an extremely short mRNAhalf-life. Further reduction could be achieved by combining 2 or moredifferent mRNA instability elements (FIG. 13). However, this is unlikelyto be required for most applications. Applications that require a moremoderate destabilising effect could utilise 1 or 2 nonamers, rather than4.

With the standard luciferase reporter, luminescence actually increasedafter the addition of doxycycline. This is most apparent when the datais expressed on a linear scale (FIG. 8) and can be explained, in part,by the delay in the action of doxycycline. However, even from 4 hrsonwards, no decay is evident, demonstrating the inadequacy of thisreporter for measuring changes in transcription over time. A furtherproblem of this vector is revealed in FIG. 10. These data relate tochanges in reporter levels over time (24-34 hrs post transfection), inthe absence of any treatment or drug. Reporter levels generally increaseduring the first 24 hrs post transfection as the plasmids enter thecells and begin to be expressed. A decrease is generally seen from about48 hrs as the plasmids are expelled from the cells. Therefore,measurements are typically taken between 24 and 48 hrs. In the absenceof drugs or treatment, the new vector (BTG1N4), containing theinstability elements, shows excellent stability of reporter levels. Incontrast, the luciferase vector is clearly still ramping up expressionlevels. Constructs with moderate stability (e.g., BTG1) showedintermediate results. Clearly reporters with longer mRNA and proteinhalf-lives will undergo a more lengthy ramping up phase as indicated inFIG. 10. The more stable expression levels seen with the new constructduring the critical period of 24-34 hrs will facilitate accuratemeasurement and represent another advantage of the invention.

The rate of decrease in reporter levels can be compared between two ormore constructs, which differ in their reporter mRNA sequence (e.g., in3′-UTR) but encode the same protein or different proteins with the samehalf-life (e.g., d2EGFP, d2EYFP). In this context, differences in therate of decay indicate an effect of the altered mRNA sequence on mRNAstability. For example, the presence of 4 UUAUUUAUU [SEQ ID NO:1]nonamers as DNA TTATTTATTT [SEQ D NO:13] (FIGS. 7-9) or the c-fos ARE(FIG. 11) [SEQ ID NO:14], within the 3′-UTR significantly increased therate of mRNA decay. In addition to demonstrating the effectiveness ofthese elements, the methods and vectors used also represent asubstantially improved system for detecting other cis-acting mRNAstability/instability elements and this process is also encompassedherein.

As shown in FIGS. 12 to 14 mRNA destabilising elements work withLuciferase, GFP and DsRed not withstanding the low level of homologybetween these reporters. DsRed has only 23% homology with EGFP. As shownin FIG. 14 myc ARE (SEQ ID NO: 21) are effective and are also effectivein combination with different destabilising elements.

Example 15 mRNA Destabilising Elements

RNA destabilising elements in accordance with the present invention canbe derived inter alia from the 3′-UTR of the following genes. In mostcases, the full-length 3′-UTR can be used. However, the U-rich and/orAU-rich elements can often be used alone.

-   -   (a) Phosphoenolpyruvate carboxykinase (PEPCK) mRNA destabilising        elements described by Laterza OF et al. Regions within 3′ half        of 3′-UTR referred to as JW6 and JW7 i.e.,        GTATGTTTAAATTATTTTTATACACTGCC        CTTTCTTACCCTTTCTTTACATAATTGAAATAGGTATCCTGACCA [SEQ ID NO:16].    -   (b) The Bicoid gene from Drosophila melanoeaster comprises a        mRNA destabilising element in first 43 nt of 3′-UTR (Surdej P.        et al) such an element can be used inter alia to destabilise        mRNA in insect cells.    -   (c) The Human Thioredoxin reductase gene (Gasdaska, J R et al).        The entire 3′-UTR. Nucleotide 1933-3690 (contains 6 AU-rich        elements). Segment containing 3 upstream AU repeats (nucleotide        1975-3360). There is also as Non-AU-rich destabilising element        at nt 1933-2014.    -   (d) Heat Stable Antigen (HSA) Gene described in Zhou, Q et al.        For example, nucleotides 1465-1625 in the 3′-UTR.

(e) Granulocyte-macrophage colony stimulating factor (GM-CSF) AREdescribed by Chyi-Ying, A et at AGUAAUAUUUAUAUAUUUAUAUUUUUAAAAUAUUUAUUUAUUUAUUUAUUUAA [SEQ ID NO:17] i.e., as DNA:AGTAATATTTATATATTTATATTTTTAAAATATTTATTTATTTATTTA TTTAA [SEQ ID NO:18].

-   -   (f) c-fos full length 3′-UTR or part thereof or ARE as defined        by Shyu et al 5′AA        AACGTTTTATTGTGTTTTTAATTTATTTATTAAGATGGATTCTCAGAT        ATTTATATTTTTTATTTTATTTTTTT3′ [SEQ ID NO:19] or by Peng, S et al        5′TTTTATTGTGTTTTTAATTTATTTATTAAGATGGATTCTCAGATATT        TATATTTTTATTTTATTTTTTTT3′ [SEQ ID NO:20].    -   (g) c-jun ARE as described by Peng, S et al.        5′UUUCGUUAACUGUGUAUGUA        CAUAUAUAUAUUUUUUAAUUUGAUUAAAGCUGAUUACUGUGAAU        AAACAGCUUCAUGCCUUUGUAAGUU3′ [SEQ ID NO:21] Sequence as DNA:        5TTTCGTTAACTGTGTATGTATGTACATATATATATTTTTTAATTTGA        TTAAAGCTGATTACTGTGAATAAACAGCTTCATGCCTTTGTAAGTT3′ [SEQ ID NO:22]        or the mutant thereof which does not contain a polyadenylation        (AAUAAA [SEQ ID NO:23]) signal i.e.,        5′UUUCGUUAACUGUGUAUGUACAUAUAUAUAUUUUUUAAUUUGA        UUAAAGCUGAUUACUGUGgAUgAUccACAGCUUCAUGCCUUUGUAAGUU 3′ [SEQ ID        NO:24] or as DNA 5′TTTCGTTAACTGTGTATGTACA        TATATATATTTTTTAATTTGATTAAAGCTGATTACTGTGgATccACAGC        TTCATGCCTTTGTAAGTT3′ [SEQ ID NO:25].    -   Sequences from the following genes, that include their        respective ARE components as described by Henics, T. et al.:    -   (h) IFN-γ ARE: 5′UCUAUUUAUUAAUAUUUAACAUUAUUUAUAUAU GGG3′ [SEQ ID        NO:26] or as DNA 5′TCTATTTATTAATATTTAAC ATATTTATATATGGG3′ [SEQ        ID NO:27].

(i) IL-2 ARE: [SEQ ID NO:28]5′CUCUAUUUAUUUAAAUAUUUAACUUUAAUUUAUUUUUGGAUGUAUUGUUUACUAACUUUUAGUGCUUCCCACUUAAAACAUAUCAGGCUUCUAUUUAUUUAAAUAUUUAAAUUUUAUAUUUAUU3′ or as DNA [SEQ ID NO:29]5′CTCTATTTATTTAAATATTTAACTTTAATTTATTTTTGGATGTATTGTTTACTAACTTTTAGTGCTTCCCACTTAAAACATATCAGGCTTCTATTTATTTAAATATTTAAATTTTATATTTATT3′

-   -   (j) c-myc ARE (see also SEQ ID NO:49): 5′AUAAACCCUAAUUUUU        UAUUUAAGUACAUUUUGCUUUUAAAGUU3′ [SEQ ID NO:30] or as DNA        5′ATAAACCCTAATTTTTTTTATTTAAGTACATTTTGCTTTTAAA GTT3′ [SEQ ID        NO:31].

(k) IL-10: [SEQ ID NO:32]5′UAGAAUAUUUAUUACCUCUGAUACCUCAACCCCCAUUUCUAUUUAUUUACUGAGCUUCUCUGUGAACGAUUUAGAAAGAAGCCCAAUAUUAUAAUUUUUUUCAAUAUUUAUUAUUUUCA3′ or as DNA [SEQ ID NO:33]5′TAGAATATTTATTACCTCTGATACCTCAACCCCCATTTCTATTTATTTACTGAGCTTCTCTGTGAACGATTTAGAAAGAAGCCCAATATTATAATTTTTTTCAATATTTATTATTTTCA3′.

-   -   (l) bcl-2: Sequences from the bcl-2 3′-UTR that include all or        part of the bcl-2 ARE as defined by Schiavone, N et al.        5′UCAGCUATWUACUGCC AAAGGGAAAUAUCAUUUAUUUUACAUUAUUAAGAAAAAAGAU        UUAUUUAUUUAAGACAGUCCCAUCAAAACUCCGUCUUUGGAAAUC 3′ [SEQ ID NO:34]        (M13994 from nt 2371-2475) or as DNA        5′TCAGCTATGCAAAGGGAAATATCATTrATTACATTAT        TAAGAAAAAAGATTTATTTATTTAAGACAGTCCCATCAAAACTCCGT CTTTGGAAATC3′        [SEQ ID NO:35].    -   (m) TNF ARE: as described by Xu, N et al. 5′-UUAUUUAUUA        UUUAUUUAUUAUUUAUUUAUUUA3′ [SEQ ID NO:36] or as DNA 5′ATTATTTATT        ATTTATTTATTATTTATTT ATTTA-3′[SEQ ID NO:37].    -   (n) IL-3 ARE: as described by Xu, N et al. 5′UAUUUUAUUCCAUU        AAGGCAUUUAUUUAUGUAUUUAUGUAUUUAUUUAUUUAUU3′ [SEQ ID NO:38] or as        DNA 5′-TATTTTATTCCATTAAGGCTATTTAT        TTATGTATTTATGTATTTATTTATTTATT-3′ [SEQ ID NO:39].    -   (o) The nonamer UUAUUUAUU [SEQ ID NO:1] as DNA TTATTTATT [SEQ ID        NO:13], as described by Zubiaga, A et al.    -   (p) The nonamer UUAUUUA(U/A)(U/A) [SEQ ID NO:2] as DNA        TTATTTA(T/A)(T/A) [SEQ ID NO:40] as described by Lagnado, C et        al.    -   (q) The pentamer AUUUA [SEQ ID NO:3] as described by Xu, N et        al. or as DNA ATTTA [SEQ ID NO:41].    -   (r) The tetramer AUUU [SEQ ID NO:4] or as DNA ATTT [SEQ ID        NO:42].    -   AU-rich elements (AREs) in general of both class I and class II        as described by Chen, C and Shyu, A.    -   (s) Plants have DST (downstream sequences) which act as        destabilising elements. DST sequence are defined in: Newnan, T        et al. A proposed consensus DST sequence is:        GGAgN₂₋₉cATAGATTaN₃₋₈(A/C)(T/A)(A/T)TttGTA(T/C) [SEQ ID NO:43].    -   This is based on comparison of 9 different DST sequences.    -   Bold=conserved in 9/9 genes.    -   Capital=conserved in at least 7/9 genes    -   N2-9=variable length region of 2-9 nucleotides; average=5.    -   N3-8=variable length region of 3-8 nucleotides; average=6.    -   Distance from stop codon=19-83 nt.

Further examples of DST sequences include the: Soybean 10A5 gene:5′GGAGN₅CATAGATTAN₈AAATTTGTAC3′. [SEQ ID NO:44] Arabidopsis SAURACIgene: 5′GGAAN₉CATAGATCGN₈CAATGCGTAT3′. [SEQ ID NO:45]

-   -   DST sequences are an alternative to AU-rich elements for use in        plants. Both AU-rich elements and DST sequences destabilise        transcripts in plants.    -   (t) Iron Responsive Element (IRE):        -   as described for example by Thomson, A et al. 1999.        -   IREs contain consensus CAGUG in a hairpin-loop.

EXAMPLES

Ferritin IRE: GUUCUUGCUUCAACAGUGUUUGAACGGAAC [SEQ ID NO:46] or as DNAGTTCTTGCTTCAACAGTGTTTGAACGGAAC. [SEQ ID NO:47] Transferrin Receptor IRE:GAUUAUCGGGAGCAGUGUCUUCCAUAAUC [SEQ ID NO:48] or as DNAGATTATCGGGAGCAGTGTCTTCCATAATC. [SEQ ID NO:49]

-   -   Iron Regulatory Proteins (IRPs; e.g., IRP1 and 2) bind IREs in        an iron-dependent fashion. Binding is also modulated by various        other stimuli and treatments (e.g., oxidative stress, nitric        oxide, erythropoietin, thyroid hormone or phosphorylation by        PKCs.    -   IREs can modulate both translational efficiency and mRNA        stability. For example, the 5′-UTR IRE in Ferritin mRNA blocks        translation only when bound to an IRP. The IREs in the 3′-UTR of        Transferrin receptor mRNA inhibit mRNA decay when bound by an        IRP. Therefore, IREs can be inserted into 5′-UTR or 3′-UTR of        expression vectors to provide expression that can be controlled        by modulating iron levels or other stimuli.    -   Destabilising elements can be used with Clontech's Mercury        Pathway Profiling vectors and in vivo kinase assay kits.        Clontech produce 3 different protein-destabilising elements, all        containing a PEST sequence and all derived from the MODC gene.        Different mutant MODCs placed at the carboxy-end of EGFP provide        protein half-lives of 1 hr, 2 hr and 4 hr. mRNA destabilising        elements in accordance with the present invention can be used in        conjunction with these and any other protein-destabilising        element (e.g., ubiquitination signals).

(u) c-myc ARE may also be defined as: [SEQ ID NO:50]5′ATGCATGATCAAATGCAACCTCACAACCTTGGCTGAGTCTTGAGACTGAAAGATTTAGCCATAATGTAAACTGCCTCAAATTGGACTTTGGGCATAAAAGAACTTTTTTATGCTTACCATCTTTTTTTTTTCTTTAACAGATTTGTATTTAAGAATTGTTTTTAAAAAATTTTAAGATTTACACAATGTTTCTCTGTAAATATTGCCATTAAATGTAAATAACTTT3′

-   -   (v) Another useful mRNA element can be obtained from histone        mRNA, Specifically, 3′-UTR sequences including a consensus stem        loop structure are described by Gallie, D et al.:        UGA-N₂₀₋₄₀-CCAAAGGYYYUUYUN ARRRCCACCCA [SEQ ID NO:51], where        Y=pyrimidine, R=purine, N=any nucleotide or as DNA        TGA-N₂₀₋₄₀-CAAAGGYYYTTYTARRRCCACCCA [SEQ ID NO:52].

Such sequences can increase translational efficiency. Moreover, they arecapable of directing mRNA decay specifically outside of S phase.Reporter constructs containing a cell-cycle-specific promoter, togetherwith mRNA destabilising elements are contemplated in this invention as atool for directing cell-cycle specific expression (e.g., of a reporter).The histone 3′-UTR element offers an alternative for use with an S-phaseor late G1 specific promoter, since it will direct increased mRNA decayin G2 relative to S-phase, thus further restricting protein expressionto S phase.

Yet another use of 3′-UTR elements in expression vectors is for thepurpose of specifically localising the chimeric mRNA. For example, theutrophin 3′-UTR is capable of directing reporter mRNA to thecytoskeletal-bound polysomes. mRNA stabilising elements are alsocontained in this 3′-UTR (Gramolini, A, et al.).

Example 16 mRNA Stabilising Elements and Expression Vectors Encoding aStabilised mRNA

Stabilising sequences may contain CT-rich elements and/or sequencesderived from long-lived mRNAs (particularly 3′-UTR regions)

CT-rich elements may contain (C/U)CCAN_(x)CCC(U/A)Py_(x)UC(C/U)CC [SEQID NO:53] as described by Holcik and Liebbaber, 1997.

CT-rich elements may contain the following elementCCTCCTGCCCGCTGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGC [SEQ ID NO:54] or partsthereof, including CCTCC [SEQ D NO:55], CCTCCTGCC [SEQ ID NO:56] orCCCTCCTCCCCTGG [SEQ ID NO:57].

A 14-nt pyrimidine-rich region from the 3′-UTR of human beta-globindescribed by Yu and Russell is also contemplated for use as astabilising element.

Examples of long-lived mRNAs from which stabilising elements may bederived include; Alpha2 globin, Alpha1 globin, beta globin. From human,mouse, rabbit or other species, bovine growth hormone 3′-UTR.

The mRNA instability elements described herein generally act in adominant-fashion to destabilise chimeric genes. It follows, thereforethat mRNA stabilising elements are often recessive-acting. For example,insertion of a c-fos ARE into the rabbit beta-globin gene, results in adestabilised transcript despite the continued presence of mRNA stabilityelements (Shyu, A et al. 1989). Both alpha- and beta-globin mRNAscontain stability elements that have been mapped to their respective3′-UTRs, whereas zeta-globin mRNA lacks these elements and is lessstable. Replacing the zeta-globin 3′-UTR with that of alpha globin mRNAnearly doubles mRNA stability (Russell, J et al. 1998). However, suchelements do not stabilise all transcripts. Therefore, the requirementsfor generating an expression vector that expresses a stable mRNA differ,dependent on the original mRNA that is to be stabilised. To create sucha vector it is generally preferable to include large segments from astable gene such as alpha- or beta-globin. With these examples, suchsegments should preferably include the entire globin 3′-UTR, replacingthe endogenous 3′-UTR. As exemplified with zeta-globin, this issometimes sufficient. However, the further incorporation ofprotein-coding and/or 5′-UTR sequences is often required. Generally, itis preferable to replace any endogenous AU- or U-rich regions, which mayact as dominant destabilising elements (these can be identified usingthe techniques described herein). Such regions in the 5′-UTR or 3′-UTRare simply replaced with alpha- or beta-globin sequences from the samerelative position. Instability elements from the coding region can berendered non-functional by mutation to synonymous codons. The globinprotein-coding region can be incorporated into the coding region of thegene of interest to create an N- or C-terminal fusion protein. Howeverthis is often not desirable and it is generally sufficient to localisethe globin protein-coding region (and 3′-UTR) into the 3′-UTR of thechimeric gene. This allows expression of the desired protein from a morestable transcript, thus markedly increasing levels of the protein. Whenthe desired protein is a reporter or is fused to a reporter or can beeasily distinguished from endogenous protein, the TRE vector systemdescribed herein (see FIG. 7) greatly facilitates the testing ofchimeric constructs for mRNA stability.

Example 17

Further Evidence of the Versatility and Effectiveness of the System

To further demonstrate the applicability of the destabilising system toa number of different reporters and applications, the inventorconstructed a wide range of reporter vectors, with and without a mRNAdestabilising element (N4; 4 copies of the nonamer TTATTTATT [SEQ IDNO:13]), and with or without a protein-destabilising element (MODC PESTsequences at carboxy-end and/or ubiquitin sequences at the N-end). TheTRE promoter was utilised, which is repressed in response to doxycyclineand slightly enhanced by PMA or a synthetic promoter comprised of 4copies of the NF-κB-binding sequence, which is more strongly enhanced byPMA. The inventor also utilised the following reporter proteins; EGFP,EYFP, ECFP, HcRed, firefly luciferase, Renilla luciferase and betagalactosidase.

Reporter constructs were assembled in the pGL3 vector backbone usingstandard cloning techniques. Essentially, the reporter genes comprised:

Sac1-promoter and 5′-UTR-Bgl2-Age1-Kozak-START-reporter proteinencoding-Hind3-MODC-PEST-encoding-STOP-Xbal-N4-SV40pA-Sal1; for thedestabilised variants and:

Sac1-promoter and 5′-UTR-Age1-Kozak-START-reporter proteinencoding-STOP-Xbal-SV40 pA-Sal1; for the standard reporter genes.

Reporter protein-encoding sequences were typically obtained by PCR ofstandard commercial vectors and cloned into the abovementioned vectorsby utilising a forward primer containing an Age1 site and a Kozaksequence and a reverse primer containing either a Hind3 site (but noSTOP codon) to create the destabilised reporter protein or an Xba1 site(downstream of the STOP codon) for the stable (standard) reporterprotein. The N4 mRNA destabilising sequence was conveniently included orexcluded from specific vectors by substituting the Xba1-Sal1 fragmentsshown above that either contain or do not contain N4. In some specificvectors, N-terminal fusions were created by inserting in frame and intothe Age1 site; a human ubiquitin sequence, preceded by a Kozak sequenceand followed by a short N-terminal destabilising element (generated byPCR of human genomic DNA) and/or the puromycin-resistance gene (withoutstop codon; generated by PCR of pBabe-puro). The Quick Change method(Invitrogen) was used to make small mutations including conversion ofthe wild-type ubiquitin sequence to a non-cleavable mutant (Gly-Valsubstitution in last amino acid residue of ubiquitin).

Nomenclature (additive to that shown in Example 14):

-   -   B (at start of name of all vectors)=Vector backbone derived from        Promega's pGL3-Basic.

Promoters:

-   -   T=Tetracycline-responsive element (RE), including the minimal        CMV promoter/5′-UTR sequence, derived from Clontech's        pTRE-d2EGFP vector.    -   N (following B)=4 tandem copies of the NFκB-binding site        followed by the minimal CMV promoter/5′-UTR.

N-terminal fusion sequences:

-   -   u=ubiquitin coding sequence followed by arginine and        destabilising N-terminal peptide.    -   mu=mutant (non-cleavable ubiquitin).    -   puro=sequence encoding puromycin-resistance.    -   neo=sequence encoding neomycin-resistance.

Parental reporter sequences:

-   -   G=EGFP coding (alone or as fusion).    -   Y=EYFP coding (alone or as fusion).    -   C=ECFP coding (alone or as fusion).    -   H=HcRed coding (alone or as fusion).    -   R=DsRed2 used as the reporter.    -   L=Firefly luciferase coding sequence (alone or as fusion).    -   Rn=Renilla luciferase coding sequence (alone or as fusion).    -   B (in middle of name)=beta-galactosidase coding sequence (alone        or as fusion).

C-terminal fusion sequences:

-   -   1=the mutated, MODC-derived, PEST sequence as present in        d1EGFP-N1 (Clontech).    -   2=the mutated, MODC-derived, PEST sequence as in pTRE-d2EGFP        (Clontech).

3′-UTR additions:

-   -   N4=4 copies of the nonamer TTATTTATT [SEQ ID NO:13] inserted        into the 3′-UTR-encoding region.

Each TRE-containing plasmid was evaluated for its rate of response (inreporter levels) to an inhibition of transcription. Briefly, each vectorin a series was transfected into Tet-Off HeLa cells, then split intomultiple dishes. Reporter levels were measured during a time-courseafter addition of doxycycline (which inhibits transcription from TRE),as described in the legends to FIGS. 15 and 17. For convenience, FIGS.15-24 all show the standard vectors as open squares on a thin line andthe fully destabilised vector as closed triangles on a bold line.

In all cases, both mRNA- and protein-destabilising elements were shownto improve the rate of response to drug (effective decay afterdoxycycline). In particular, the combination of the d1 MODC proteindegradation signal plus the N4 mRNA-degradation signal (i.e., vectorsending in 1N4) resulted in a very rapidly responding Renilla luciferase(FIG. 15), firefly luciferase (FIG. 16), EGFP (FIG. 17), EYFP (FIG. 18),ECFP (FIG. 19) and HcRed (FIG. 20). The further addition of anN-terminal ubiquitin sequence resulted in further destabilisation ofEYFP (FIG. 22B) and beta galactosidase (FIG. 21).

In theory, a more rapidly degrading reporter system such as thatdescribed herein, should not only produce faster decay after inhibitionbut should produce a more rapid accumulation after activation. Indeed,given that most drugs and biological systems involve a transient ratherthan permanent increase or decrease in expression, the more rapidresponse of unstable reporters should also lead to a larger maximumeffect in such systems. FIG. 24 shows that the destabilised Renillaluciferase does indeed show a much larger as well as more rapid increasein reporter levels following activation by PMA. The inventor has alsoused kinetic models to quantify this effect and showed that the accuratedetection of minor or transient changes is virtually impossible withmany standard reporters such as luciferases, beta galactosidase and CAT.This theoretical evidence was demonstrated in practice through theexperiment depicted in FIG. 23, which shows virtually no change instandard luciferase reporter levels after a small to moderate inductionof transcription, whereas a 4-5 fold increase was seen with thedestabilised counterpart.

Example 18 Dual-Reporter Vectors for Studying or Measuring GeneRegulation

Dual-reporter bi-directional vectors based on the example shown in FIG.4B were constructed using standard techniques and using BTH1N4 andBTG1N4 as starting material. In these dual-colour vectors, a single TREpromoter drives transcription of destabilised HcRed in one direction(BTH1N4) and destabilised EGFP in the other (BTG1N4). Convenient uniquecloning sites were introduced on the EGFP side at the transcriptionstart site and immediately upstream of the Kozak sequence. Using thesecloning sites, a variety of different 5′-UTRs were cloned into theBTG1N4 mRNA encoding region, with the BTH1N4 mRNA encoding regionremaining unchanged. As such, red fluorescence serves as an internalcontrol for transfection efficiency, cellular conditions etc. In anadditional construct, the EGFP-coding region (from BTG1N4) was fusedwith the coding region from the puromycin resistance gene to create apuro-GFP fusion protein construct (BTpuroG1N4).

Each construct was transfected into Tet-Off HeLa cells and 24 hrs later,green and red fluorescence was measured simultaneously by flow cytometryand analysed using FlowJo software. When fluorescence was expressed asthe relative ratio of green:red fluorescence (FIG. 28A), the effect ofthese different 5′-UTRs (or puro-GFP fusion protein) on expressionlevels of GFP could be easily seen. The higher relative expression ofGFP in constructs containing the Hsp70 and beta-globin 5′-UTRs isconsistent with reports of these UTRs containing translational enhancersequences. The synthetic 5′-UTR sequence showed an apparently lowtranslational efficiency. The puro-GFP construct appeared to be veryefficiently translated and this shows how the vector system can be usedto assay the effect on expression levels of protein-coding sequences aswell as UTR sequences.

Increased expression caused by a 5′-UTR (or other sequence) could derivenot only from increased translational efficiency but also from increasedmRNA stability or enhancement of transcription. An important feature ofthe embodiment exemplified here is the ability to distinguish thesepossibilities. A transcriptional enhancer, by definition, actsindependently of orientation and if present in a sample 5′-UTR, wouldenhance transcription from the TRE in both directions and thus increaseboth red and green fluorescence. A co-transfected control vector wouldassist in identifying such transcriptional enhancers, which would not beexpected to alter the green:red ratio in the manner shown in FIG. 28A.An effect on mRNA stability is a likely consequence of altered 5′-UTRsequence and with standard reporter systems this mRNA stability effectcannot be distinguished from a translational effect. However, FIG. 28Bshows that all 6 constructs have similar rates of decay in GFPfluorescence after doxycycline was added to the cells expressing theseconstructs, in order to block transcription from the TRE promoter.Therefore, it can be inferred that the different 5′-UTR sequences didnot affect mRNA stability and instead must have altered translationalefficiency.

Those skilled in the art will be aware that the invention describedherein is subject to variations and modifications other than thosespecifically described. It is to be understood that the inventiondescribed herein includes all such variations and modifications. Theinvention also includes all such steps, features, compositions andcompounds referred to or indicated in this specification, individuallyor collectively, and any and all combinations of any two or more of saidsteps or features.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. Those of skill in the artwill therefore appreciate that, in light of the instant disclosure,various modifications and changes can be made in the particularembodiments exemplified without departing from the scope of the presentinvention. All such modifications and changes are intended to beincluded within the scope of the appended claims. TABLE 1 Signaltransducers that could be used in the present invention Signaltransducer AKT (also called PKB) Fas L/BID JAK 7 Stat MKK-47/JNKMTOR/p70 s6 kinase NFκB p38 PKA/Rap1 B-raf Ras/Raf Wnt/GSK3 Erk 1&2

BIBLIOGRAPHY

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1. A method for assaying the activity of a transcriptional controlelement, the method comprising: expressing from the transcriptionalcontrol element a polynucleotide that encodes a polypeptide and that isoperably connected to a nucleic acid sequence that encodes a RNA elementthat modulates the stability of a transcript encoded by thepolynucleotide; and measuring the level or functional activity of thepolypeptide produced from the expression.
 2. A method according to claim1, wherein the RNA element is a destabilising element which reduces thestability of the transcript.
 3. A method according to claim 1, whereinthe polynucleotide and the nucleic acid sequence are heterologous toeach other.
 4. A method according to claim 1, wherein the polypeptidehas an intracellular half-life of less than about 3 hours.
 5. A methodaccording to claim 1, wherein the polypeptide comprises aprotein-destabilising element.
 6. A method according to claim 5, whereinthe protein-destabilising element is selected from a PEST sequence, anN-terminal destabilising amino acid or an ubiquitin or a biologicallyactive fragment thereof, or variant or derivative of these.
 7. A methodaccording to claim 1, wherein the polypeptide is a reporter protein. 8.A method according to claim 7, wherein the reporter protein is selectedfrom an enzymatic protein or a protein associated with the emission oflight.
 9. A method according to claim 7, wherein the reporter protein isa fluorescent protein or a luminescent protein.
 10. A method accordingto claim 1, wherein the expression of the polynucleotide is carried outin the presence of a test agent.
 11. A method according to claim 10,wherein the method further comprises: comparing the level or functionalactivity of the polypeptide produced in the presence and absence of thetest agent.
 12. A method according to claim 10, wherein the expressionof the polynucleotide is carried out in a first cell type or conditionand in a second cell type or condition, wherein a difference in thelevel or functional activity of the polypeptide in the presence of thetest agent between the cell types or conditions provides information onthe effect of the test agent on the cell types or conditions.
 13. Amethod according to claim 1, wherein the method comprises: expressingfrom the first transcriptional control element in a first construct afirst polynucleotide that encodes a first polypeptide and that isoperably connected to a nucleic acid sequence that encodes a RNA elementthat modulates the stability of a transcript encoded by the firstpolynucleotide; measuring the level or functional activity of the firstpolypeptide produced from the first construct; expressing from a secondtranscriptional control element in a second construct a secondpolynucleotide that encodes a second polypeptide and that is operablyconnected to a nucleic acid sequence that encodes a RNA element thatmodulates the stability of a transcript encoded by the secondpolynucleotide, wherein the expression of the second polynucleotide iscarried out in the presence or absence of the test agent, and whereinthe second transcriptional control element is different than the firsttranscriptional control element; measuring the level or fiinctionalactivity of the second polypeptide produced from the second construct;and comparing the level or functional activity of the second polypeptidewith the level or functional activity of the first polypeptide in thepresence or absence of the test agent.
 14. A method according to claim13, wherein the first construct and the second construct are bothpresent on a single vector.
 15. A method according to claim 13, whereinthe first construct and the second construct are present on differentvectors.
 16. A method according to claim 13, wherein the firstpolypeptide and the second polypeptide are detectably distinguishable.17. A method according to claim 13, wherein the first construct and thesecond construct are contained within a single cell.
 18. A methodaccording to claim 13, wherein the first construct and the secondconstruct are contained within different cells.
 19. A method accordingto claim 13, wherein at least one of the first and second polypeptideshas an intracellular half-life of less than about 3 hours.
 20. A methodaccording to claim 13, wherein both the first and second polypeptideshave an intracellular half-life of less than about 3 hours.
 21. A methodaccording to claim 1, wherein the activity of the transcriptionalcontrol element is a measure of a cellular event.
 22. A method accordingto claim 21, wherein the cellular event is selected from cell cycleprogression, apoptosis, immune function, modulation of a signaltransduction pathway, modulation of a regulatory pathway, modulation ofa biosynthetic pathway, toxic response, cell differentiation and cellproliferation.